Phospholipid derivatives of non-steroidal anti-inflammatory drugs

ABSTRACT

Disclosed are compounds having non-steroidal anti-inflammatory drugs (NSAIDS) covalently linked to a phospholipid moiety via a bridging group. Also disclosed are a process for the synthesis of the compounds, pharmaceutical compositions comprising the compounds and the use thereof for the treatment of diseases and disorders related to inflammatory conditions, such as the treatment of ischemia.

FIELD OF THE INVENTION

[0001] The present invention relates to compounds comprisingnon-steroidal anti-inflammatory drugs (NSAIDs) covalently linked to aphospholipid moiety via a bridging group, to pharmaceutical compositionscomprising such compounds and to the use thereof for the treatment ofdiseases and disorders related to inflammatory conditions. The inventionfurther relates to a process for the synthesis of said phospholipidderivatives of NSAIDs.

BACKGROUND OF THE INVENTION

[0002] Inflammation is an important aspect of the natural defenseprocess. Inflammation becomes a pathological process, requiring medicalintervention, when inflammatory mediators cause excessive damage to thesurrounding tissue. Examples of such pathological processes arerheumatoid arthritis (RA) and psoriasis. Recently, a significantinflammatory component has been found in other types of disease, forexample, neurological disorders such as multiple sclerosis andAlzheimer's disease. A common feature of many inflammatory diseases isan elevation in phospholipase A₂ (PLA₂) activity.

[0003] PLA₂ is the common name for a diverse group of enzymes thatspecifically hydrolyze the sn-2 bond of glycerophospholipid to releasefree fatty acids and lysophospholipids. PLA₂ is thought to be ratelimiting in the release of arachidonic acid. The other product of itsreaction, lysophospholipid, is thought to be the precursor of plateletactivating factor (PAF). PAF and the arachidonic acid metabolites,eicosanoids, are pro-inflammatory lipid intermediates derived frommobilized cell membrane phospholipids by the action of phospholipaseenzymes. PLA₂ is thus implicated as having a crucial role in theproduction of the entire cascade of phospholipid-derived inflammatorymediators.

[0004] For persistent inflammation three classes of drugs are widelyused, corticosteroids, non-steroidal anti-inflammatory drugs (NSAIDs)and slow acting disease modifying drugs.

[0005] Corticosteroids are the most potent and effective agents incontrolling inflammatory conditions. Unfortunately, prolonged use ofthese drugs is associated with side effects. Topical corticosteroidpreparations are widely used for inflammatory dermatological conditionsand inhaled corticosteroids account for 55% of the asthma market in theUnited Kingdom. Prednisolone is the most commonly administeredcorticosteroid in RA, which though possibly affecting the underlyingdisease process does not provide a cure and is associated with severeside-effects.

[0006] NSAIDs relieve the symptoms of inflammation without altering thecourse of the disease, but they have adverse gastrointestinal and renalside effects. Their main action is inhibition of arachidonatecyclooxygenase (in inflammatory cells the COX₂ isoenzyme) and thusinhibiting prostaglandin and thromboxane production.

[0007] Disease-modifying anti-rheumatic drugs (DMARDs) such as goldproducts, auranofin (SKB), chloroquine (Sanofi), sulfasalazine(Pharmacia & Upjohn), cyclosporin (Sandoz (Novartis)) and methotrexate(MTX, APH, Pharmacia & Upjohn) are second-line agents for treatment ofRA. In most cases their mode of action is ill defined and the term‘slow-acting’ is applied because these agents may take weeks or monthsto have demonstrable effect. Treatment with DMARDs has to be continuedfor years. If complete remission is achieved for at least six months,the dosage is gradually reduced and may be stopped altogether. DMARDsappear to decrease radiographic joint damage and improve acute-phasemarkers in RA but they all have adverse effects.

[0008] Diclofenac (o-[(2,6-dichlorophenyl)amino]phenylacetate) is anon-steroidal anti-inflammatory drug of the phenylacetic acid class.When given orally the absorption of diclofenac is rapid and complete. Itbinds extensively to plasma albumin. Substantial concentrations of drugare attained in synovial fluid, which is the proposed site of action ofthe NSAIDs. Diclofenac is a potent inhibitor of prostaglandin synthesisand has also been shown to inhibit interleukin-1 (IL-1β) and tumornecrosis factor alpha (TNF-α), involved in osteoarthritis.Gastrointestinal complications such as ulceration and intolerance arethe most common adverse effect of diclofenac. Renal dysfunction andhypersensitivity reactions also occur. Many patients with rheumaticdisorders have some degree of renal function impairment and areespecially susceptible to the induction of renal failure by NSAIDs.

[0009] Other non-steroidal anti-inflammatory drugs such as salicylates,indomethacin and ibuprofen directly inhibit cyclooxygenase, a key enzymein the synthesis pathway of prostaglandins. However, since these drugsinhibit early reactions in the arachidonic acid metabolism, they mayblock the formation of more than one product, hence leading to severeside effects. Indomethacin, for example, may also disrupt calcium fluxacross membranes, inhibit cAMP-dependent protein kinase andphosphodiesterase.

[0010] It would, therefore, be desirable to be able to extend theusefulness of NSAIDs to conditions that do not respond to lower doses ofthe drugs and to reduce undesirable side effects by their targeting tothe diseased cells.

[0011] The use of prodrugs to impart desired characteristics such asincreased bioavailability or increased site-specificity for known drugsis a recognized concept in the state of the art of pharmaceuticaldevelopment. The use of various lipids in the preparation of particulartypes of prodrugs is also known in the background art. However, none ofthe background art discloses prodrugs comprising NSAIDs that uponactivation by intracellular lipases enable preferential accumulation andrelease of the drug within the diseased cells.

[0012] International Patent Application WO 91/16920 disclosesphospholipid prodrugs of salicylates and nonsteroidal anti-inflammatorydrugs wherein the drug is directly linked, without any spacer, to eitheror both of the glycerol hydroxyls of a phospholipid or to availablehydroxyls or amines of phospholipid head groups. These prodrugs, whentaken orally, protect the gastric mucosa and release the activeprinciple in the gut via the action of pancreatic enzymes.

[0013] In other examples of phospholipid prodrugs, the formulation ofthe prodrugs into liposomes or other micellar structures is the featurethat enables their preferential uptake, for instance by liver cells orby macrophages as in the case of the phospholipid conjugates ofantiviral drugs disclosed in International Patent Applications WO93/00910 and WO 90/00555.

[0014] International Patent Application WO 96/22780 disclosescompositions comprising nonsteroidal anti-inflammatory drugsnon-covalently associated with zwitterionic phospholipids. In contrast,the present invention relates to nonsteroidal anti-inflammatory drugscovalently bound to a phospholipid via a spacer group.

[0015] U.S. Pat. No. 5,149,794 discloses a method for delivering drugsselectively to intracellular organelles. The disclosed compoundscomprise an antiviral or antineoplastic drug covalently bound to a lipidcarrier via a spacer group which may act to modulate drug release at thetarget site. In contrast to the present invention, the disclosed prodrugis site specific due to the existence of the lipid carrier, and drugrelease from the lipid conjugate is not a requirement for the drugtargeting. In addition, said U.S patent does not disclose phospholipidsas the lipid carriers, nor compounds comprising nonsteroidalanti-inflammatory drugs.

[0016] U.S. Pat. No. 5,256,641 discloses methods of delivering andspecifically targeting antigenically-active peptides to cells for thespecific production of immunological reactivity against such peptides.In contrast, the present invention does not disclose prodrugs whereinthe active ingredients are peptides, though peptides may serve as aspacer between the active drug and the phospholipid.

[0017] U.S. Pat. No. 5,543,389 discloses covalent polar lipid-drugconjugates for facilitating the entry of drugs into cells atpharmokinetically useful levels. The rationale for specific activationof the prodrug in that case, are very different from the presentinvention. The examples of the present invention with phospholipids werenot made and with hindsight it is clear that it would be ineffective tosynthesize active phospholipid prodrugs if the spacer between the lipidand the drug is less than a specific length of at least 4 carbon atoms,because of unfavorable conditions due to steric hindrance andstereochemical problems.

SUMMARY OF THE INVENTION

[0018] The present invention provides, in one aspect, compounds of thegeneral formula I

[0019] or a pharmaceutically acceptable salt thereof, wherein:

[0020] R1 is a saturated or unsaturated, substituted or unsubstitutedhydrocarbon chain having from 2 to 30 carbon atoms;

[0021] R2 is H or a phospholipid head group;

[0022] D is the residue of a nonsteroidal anti-inflammatory drug havinga functional group selected from the group consisting of carboxyl,hydroxyl, amine and thiol, wherein D is attached through said functionalgroup to a bridging group, —C(O)-Z-X—, wherein Z is a saturated orunsaturated hydrocarbon chain having from 2 to 15 carbon atoms, and X isselected from amino, hydroxy, thio and carbonyl groups, such that whenthe functional group of D is carboxyl, X is selected from amino, hydroxyand thio, and when the functional group of D is amino, hydroxy or thio,X is a carbonyl group.

[0023] In one preferred embodiment, R1 of the above compound of formulaI is a hydrocarbon chain having from 10 to 20 carbon atoms, preferablyan alkyl residue of 15 or 17 carbon atoms.

[0024] According to another preferred embodiment, the nonsteroidalanti-inflammatory drug is selected from the group including, but notlimited to, diclofenac, indomethacin, ibuprofen, naproxen and6-methoxy-2-naphthylacetic acid.

[0025] In still another preferred embodiment, the phospholipid headgroup R2 is selected from choline, ethanolamine, inositol and serine.

[0026] Preferred according to the invention are compounds of the generalformula I wherein the drug residue D is inactive while bound to the—C(O)-Z-X— bridging group and the release of the active drug isinitiated by enzymatic cleavage of an ester bond at position sn-2 of thephosholipid. Preferably the enzymatic cleavage is executed by aphospholipase, more preferably phospholipase A₂ (PLA₂).

[0027] Most preferred compounds according to the invention are:

[0028]1-Stearoyl-2-{3-[2′-(2″,6″-dichloroanilino)phenylacetamido]propanoyl}-sn-glycero-3-phosphatidylcholine,

[0029]1-Stearoyl-2-{4-[2′-(2″,6″-dichloroanilino)phenylacetamido]butanoyl}-sn-glycero-3-phosphatidylcholine,

[0030]1-Stearoyl-2-{5-[2′-(2″,6″-dichloroanilino)phenylacetamido]valeroyl}-sn-glycero-3-phosphatidylcholine,

[0031]1-Stearoyl-2-{6-[2′-(2″,6″-dichloroanilino)phenylacetamido]hexanoyl}-sn-glycero-3-phosphatidylcholine,

[0032] 1-Stearoyl-2-{8-[2′-(2″,6″-dichloroanilino)phenylacetamido]octanoyl}-sn-glycero-3-phosphatidylcholine,

[0033]1-Stearoyl-2-{12-[2′-(2″,6″-dichloroanilino)phenylacetamido]dodecanoyl}-sn-glycero-3-phosphatidylcholine,

[0034] 1-Stearoyl-2-{3-[1-(p-chlorobenzoyl)-5-methoxy-2-methylindolylacetamido]propanoyl}-sn-glycero-3-phosphatidylcholine,

[0035] 1-Stearoyl-2-{4-[1-(p-chlorobenzoyl)-5-methoxy-2-methylindolylacetamido]butanoyl}-sn-glycero-3-phosphatidylcholine,

[0036] 1-Stearoyl-2-{5-[1-(p-chlorobenzoyl)-5-methoxy-2-methylindolylacetamido]valeroyl}-sn-glycero-3-phosphatidylcholine,

[0037] 1-Stearoyl-2-{6-[1-(p-chlorobenzoyl)-5-methoxy-2-methylindolylacetamido]hexanoyl}-sn-glycero-3-phosphatidylcholine,

[0038] 1-Stearoyl-2-{8-[1-(p-chlorobenzoyl)-5-methoxy-2-methylindolylacetamido]octanoyl}-sn-glycero-3-phosphatidylcholine,

[0039]1-Stearoyl-2-{3-[α-methyl-4-(2-methylpropyl)benzeneacetamido]propanoyl}-sn-glycero-3-phosphatidylcholine,

[0040]1-Stearoyl-2-{6-[α-methyl-4-(2-methylpropyl)benzeneacetamido]hexanoyl}-sn-glycero-3-phosphatidylcholine,

[0041]1-Stearoyl-2-{3-[(S)-6-methoxy-α-methyl-2-naphtaleneacetamido]propanoyl}-sn-glycero-3-phosphatidylcholine,

[0042]1-Stearoyl-2-{4-[(S)-6-methoxy-α-methyl-2-naphtaleneacetamido]butanoyl}-sn-glycero-3-phosphatidylcholine,

[0043]1-Stearoyl-2-{6-[(S)-6-methoxy-α-methyl-2-naphtaleneacetamido]hexanoyl}-sn-glycero-3-phosphatidylcholine,and

[0044]1-Stearoyl-2-{4-[2-(6-methoxynaphtyl)acetamido]butanoyl}-sn-glycero-3-phosphatidylcholine.

[0045] Compounds of the invention are useful for the treatment ofdiseases and disorders related to inflammatory conditions. Thesecompounds may serve as prodrugs that upon initial enzymatic cleavage,that may or may not be followed by further enzymatic or non-enzymaticcleavage(s), release nonsteroidal anti-inflammatory agents at thediseased site.

[0046] Thus, in another aspect, the invention provides pharmaceuticalcompositions comprising a pharmaceutically acceptable carrier and, as anactive ingredient, a compound of the general formula I

[0047] or a pharmaceutically acceptable salt thereof, wherein:

[0048] R1 is a saturated or unsaturated, substituted or unsubstitutedhydrocarbon chain having from 2 to 30 carbon atoms;

[0049] R2 is H or a phospholipid head group;

[0050] D is the residue of a nonsteroidal anti-inflammatory drug havinga functional group selected from the group consisting of carboxyl,hydroxyl, amine and thiol, wherein D is attached through said functionalgroup to a bridging group, —C(O)-Z-X—, wherein Z is a saturated orunsaturated hydrocarbon chain having from 3 to 15 carbon atoms, and X isselected from amino, hydroxy, thio and carbonyl groups, such that whenthe functional group of D is carboxyl, X is selected from amino, hydroxyand thio, and when the functional group of D is amino, hydroxy or thio,X is a carbonyl group.

[0051] The pharmaceutical compositions of the invention are useful forthe treatment of diseases and disorders related to inflammatoryconditions including, but not being limited to, autoimmune diseases suchas arthritis, rheumatoid arthritis, multiple sclerosis, systemic lupuserythematosus, other diseases such as asthma, psoriasis, inflammatorybowel syndrome, neurological degenerative diseases such as Alzheimer'sdisease, Parkinson's disease, Huntington's disease, vascular dementia,and other pathological conditions such as epilepsy, migraines, strokeand trauma.

[0052] Any suitable mode of administration of the pharmaceuticalcomposition can be used according to the invention including, but notbeing limited to, oral, ocular, nasal, parenteral, topical or rectaladministration. The pharmaceutical compositions may be in the form ofsolutions, suspensions, capsules, tablets, aerosols, gels, ointments orsuppositories.

[0053] In yet another aspect, the invention provides a method fortreatment of a disease or disorder related to an inflammatory conditioncomprising administering to a patient in need thereof a therapeuticallyeffective amount of a compound or a pharmaceutical composition inaccordance with the invention.

[0054] In still another aspect, the present invention provides a processfor the synthesis of compounds in accordance with the invention. Saidsynthesis process comprising:

[0055] (i) providing a molecule y-X-Z-COOH, wherein y is selected from Hand OH, Z is a saturated or unsaturated hydrocarbon chain having from 2to carbon atoms, and X is selected from amino, hydroxy, thio andcarbonyl groups;

[0056] (ii) replacing y with an appropriate blocking group, B;

[0057] (iii) preparing an anhydride of the molecule B-X-Z-COOH;

[0058] (iv) acylating a lyso-lecithin by the anhydride of step (iii) toyield 1-acyl-2-acyl(X-B)-sn-glycero-3 phospholipid;

[0059] (v) removing the blocking group B from the functional group X;and

[0060] (vi) coupling a nonsteroidal anti-inflammatory drug D to thefunctional group X,

[0061] thus, generating a molecule of the general Formula I.

[0062] Further objects of the present invention will become apparent tothose skilled in the art upon further review of the followingdisclosure, including the detailed descriptions of specific embodimentsof the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

[0063] The invention will be understood and appreciated more fully fromthe detailed description below, in conjunction with the drawings, inwhich:

[0064]FIG. 1 depicts body weight changes (%) of rats treated withmultiple per os gavage of either vehicle (open circles), 10 mg/kgdiclofenac (DCF, filled circles), 30 mg/kg1-Stearoyl-2-{4-[2′-(2″,6″-dichloroanilino)phenylacetamido]butanoyl}-sn-glycero-3-phosphatidylcholine(denoted DP-DCF; Z=3; filled squares) or 30 mg/kg1-Stearoyl-2-{6-[2′-(2″,6″-dichloroanilino)phenylacetamido]hexanoyl}-sn-glycero-3-phosphatidylcholine(denoted DP-DCF; Z=5; filled triangles).

[0065]FIG. 2 depicts gastro-toxicity scores of rats after 14-daytreatment with daily per os gavage of either vehicle (white bar), 10mg/kg DCF (striped bar), 30 mg/kg DP-DCF; Z=3 (gray bar) or 30 mg/kgDP-DCF; Z=5 (black bar).

[0066]FIG. 3 depicts effects of DCF (10 mg/kg) and DP-DCF; Z=5 (30mg/kg) on Carrageenan-induced paw oedema in rats. The treated animals'paw volumes were measured at 3, 5 and 7 hours post-Carrageenan injection(white, gray and black bars, respectively) and are presented aspercentage increase of paw volume. Statistically significant effects(p<0.05) are indicated by asterisks (*).

[0067]FIG. 4 depicts levels of neuron specific enolase (NSE) measured inthe serum of Mongolian gerbils 24 hours following induction of globalforebrain ischemia. The gerbils were treated by single oraladministration of 10 mg/kg DCF (striped bar), 30 mg/kg DP-DCF; Z=5(black bar) or vehicle (gray bar) two hours prior to induction ofischemia. NSE level in sham operated animals is presented by a whitebar. Statistically significant effect (p<0.05) is indicated by anasterisk (*).

[0068]FIG. 5 depicts levels of neuron specific enolase (NSE) measured inthe serum of Mongolian gerbils 24 hours following induction of globalforebrain ischemia. The gerbils were treated by daily oraladministration of 30 mg/kg DP-DCF; Z=5 (black bar) or vehicle (gray bar)for 5 days, beginning 4 days prior to induction of ischemia. NSE levelin sham operated animals is presented by a white bar.

DETAILED DESCRIPTION OF THE INVENTION

[0069] The present invention relates to a group of novel compoundscomprising nonsteroidal anti-inflammatory drugs (NSAIDs) covalentlyconjugated, via a bridging group, to position sn-2 of a phospholipid.

[0070] The compounds according to the invention are phospholipidderivatives wherein the conjugated NSAID residue is pharmacologicallyinactive, and regulated release of the active drug occurs at the site ofa diseased tissue. The compounds, being hydrophobic in nature, maypenetrate biological membranes and barriers, thus effectivelytransporting the attached prodrug into cells or organs. The specificityof the activation of the anti-inflammatory prodrug is afforded by thebridging group that is designed to be sensitive to cleavage byphospholipases (e.g. PLA₂) that are specifically elevated at the diseasesite. Hence, accumulation of the active drug occurs at the site of thedisease, whereas, in healthy tissue there will be only a basal level ofprodrug cleavage.

[0071] It should be appreciated that the novel compounds of theinvention wherein the NSAIDs are introduced as prodrugs, are moreeffective than their corresponding free drugs in at least two aspects:(i) having increased therapeutic efficacy at relatively lower doses, and(ii) exhibiting reduced side effects and toxicity.

[0072] Accordingly, it is possible to extend the usefulness of NSAIDs toconditions that do not respond to lower doses of the drug and to reduceundesirable side effects by the regulated release of the active drug atthe diseased site. According to a preferred embodiment, the releaseddrug is identical to the corresponding original drug used for thesynthesis of a compound of the invention, hence it is expected to have asimilar breadth of therapeutic activity. Also useful are drugderivatives that although released from the phospholipid molecule,remain conjugated to the whole or part of the bridging group, whilestill capable of exerting therapeutic effects comparable to those of theoriginal drug D.

[0073] The compounds of the invention are of the general formula I

[0074] or a pharmaceutically acceptable salt thereof, wherein:

[0075] R1 is a saturated or unsaturated, substituted or unsubstitutedhydrocarbon chain having from 2 to 30 carbon atoms;

[0076] R2 is H or a phospholipid head group;

[0077] D is the residue of a nonsteroidal anti-inflammatory drug havinga functional group selected from the group consisting of carboxyl,hydroxyl, amine and thiol, wherein D is attached through said functionalgroup to a bridging group, —C(O)-Z-X—, wherein Z is a saturated orunsaturated hydrocarbon chain having from 2 to 15 carbon atoms, and X isselected from amino, hydroxy, thio and carbonyl groups, such that whenthe functional group of D is carboxyl, X is selected from amino hydroxyand thio, and when the functional group of D is amino, hydroxy or thio,X is a carbonyl group.

[0078] It is desired that the generated covalent bond between thebridging group and the drug derivative, i.e. the X-D bond, is a stableester-, amide- or thioester bond that does not dissociate spontaneouslyunder physiological conditions.

[0079] In designing a compound according to the invention, to be used asa prodrug, the specific nature of the inflammatory condition to betreated should be considered. This involves determining the desiredpharmacological activity to be achieved hence the choice of the drug D,and identifying the particular site where the desired pharmacologicalactivity is needed.

[0080] It should be noted that the number of carbon atoms in said R1chain of a compound of the general formula I, is determined according tothe desired lipophilicity of the molecule. The lipophilicity of themolecule is directly correlated to the selected hydrocarbon chainlength. R1 chains according to the invention may contain 2 to 30 carbonatoms. Molecules with R1 having from 10 to 20 carbon atoms are mostdesired as endowing the molecule with suitable hydrophobicity forcrossing biological membranes and at the same time providing adequatesubstrate for the action of the phospholipase.

[0081] R1 may be a saturated or unsaturated hydrocarbon chain,containing one or more double bonds. One or more hydrogen atoms on thechain may be substituted, for example, by halogen atoms or by a smallalkyl group such as methyl residues, with the proviso that thesubstituents still allow free access for the desired cleaving enzymes.

[0082] In preferred embodiments of the invention R1 is an alkyl residueof an odd number of carbon atoms. More preferably R1 is an alkyl residueof 15 or 17 carbon atoms yielding, respectively, the naturally occurringpalmitoyl (C₁₆) or stearoyl (C₁₈) residues at the α position of thephospholipid.

[0083] The selection of the anti-inflammatory drug residue D to beincluded in the compound of the general formula I is dependent on thedisease or disorder intended to be treated. Any nonsteroidalanti-inflammatory drug that possesses a free —C(O)OH, —OH, —NH₂, —NH or—SH group available for reaction with the functional group of thebridging group to form a stable covalent bond D-X, may be selected.Suitable NSAIDs include, but not limited to, drugs presently known onthe market, for instance:

[0084] 1) arylacetic acid derivatives such as diclofenac, etodolac,ibufenac and indomethacin, 2) arylcarboxylic acid derivatives such asketorolac, 3) aminoarylcarboxylic acid derivatives such as flufenamicacid, meclofenamic acid, mefenamic acid and niflumic acid, 4)arylpropionic acid derivatives such as fenoprofen, ibuprofen, ketoprofenand naproxen, 5) salicylic acid derivatives such as fendosal, mesalamineand salsalate, and 6) thiazinecarboxamides such as piroxicam andtenoxicam.

[0085] Also qualified as D are active metabolites or derivatives ofNSAIDs that preserve their anti-inflammatory activity provided that theyhave an available functional group, as mentioned above, readilyavailable for reacting with the appropriate bridging group. A particularexample for this kind of drug is the compound 6-methoxy-2-naphthylaceticacid which is an in vivo metabolite of nabumetone[4-(6-methoxy-2-naphthyl)-butan-2-one].

[0086] Currently preferred embodiments according to the invention areselected from, but are not limited to, prodrugs wherein D is a residueof diclofenac, indomethacin, ibuprofen, naproxen and6-methoxy-2-naphthylacetic acid. However, any presently knownnonsteroidal anti-inflammatory drugs or those that will be available inthe future are included within the scope and concept of the presentinvention provided that said drugs contain either a carboxyl, hydroxyl,primary or secondary amine or thiol group available for participating inthe covalent bond with component X of the bridging group.

[0087] It is desirable to provide a compound of the general formula Iwherein the anti-inflammatory drug is capable of specifically inhibitingan enzyme which plays a central role in evoking inflammatory processesleading to a disease or disorder, while having no deleterious effects onother basic processes of the cell. Such desirable drugs are, forexample, Celecoxib (Searle & Co.) and Meloxicam (Boehringer, Ingelheim)which are capable of differentially inhibiting the enzyme cyclooxygenase2 (COX 2) induced in inflammatory cells, and not cyclooxygenase 1 (COX1)which is involved in normal homeostasis.

[0088] The choice of the preferred bridging group, —C(O)-Z-X—, isdependent on several considerations; it should participate in a stablecovalent bond with the D moiety while lending itself to cleavage at thetarget site. A preferred bridging group is such that is resistant tocleavage under normal physiological conditions encountered by theadministered compound on its way to the target site. The bridging groupshould not confer a steric hindrance on the enzymatic cleavage of theester bond at position sn-2 of the phospholipid of the general formulaI.

[0089] Depending on the treated inflammatory condition and theparticular diseased cell or organs, it will be desirable at times tochoose such a bridging group that will regulate the release of theactive drug by facilitating or delaying its cleavage from the prodrugmolecule.

[0090] According to a preferred embodiment, the total number of carbonatoms in the bridging group C(O)-Z-X is at least 6 but at most 15. Itwas found that this length of carbon chain provides a spacer whichenables good access to an enzyme, preferably phospholipase and inparticular PLA₂, for digesting the ester bond at position sn-2 of thephospholipid of the general formula I. Shorter spacers, in particularbridging groups comprising less than four carbon atoms, may beproblematic, by creating an unfavorable steric environment for theaction of the phospholipase. A situation of steric interference may alsobe generated by long spacers, i.e. when the number of carbon atoms inthe bridging chain is greater than 15.

[0091] X may be selected from amino, hydroxy, thio and carbonyl groupswith the proviso that when the functional group of D is —C(O)OH, X isnot a carbonyl, and when the functional group of D is —OH, —NH₂, —NH or—SH, X is a carbonyl group.

[0092] Some combinations of X with particular drugs may be unfavorableas yielding a very labile bond which is spontaneously cleaved, thereforegreatly lowering the efficacy of the prodrug. Such an unfavorablecombination is, for instance, when the functional group of D is acarboxyl group, such as, for example, in diclofenac, forming a covalentbond with X which is a carbonyl group. The resulted bond —(CO)—O—(CO)—is a labile bond that tends to dissociate.

[0093] The therapeutic efficacy of any particular compound according tothe invention should be evaluated by a person skilled in the artconsidering the general knowledge in pharmacology and the teachings ofthe present invention. The choice of a specific compound to be used as aprodrug according to the invention will also depend on the particulardisease or disorder to be treated.

[0094] It is suggested that the release of the active drug at the targetsite is initiated by a first cleavage of the compound at position sn-2of the phospholipid, preferably by a phospholipase, more preferablyphospholipase A₂. PLA₂ is the more preferred cleaving enzyme for twocompelling reasons; (i) its enhanced activity is a common feature inmany inflammatory processes and (ii) it is abnormally elevated duringthe progression of the inflammatory disease. Thus, the drug linked tothe lipid will preferentially be released at the site of theinflammation due to the increased PLA₂ activity. In accordance with theinvention, the phospholipid-NSAID conjugate prodrug is designed to haveseveral distinct advantages over the parent drug, including improvedefficacy, potency and pharmacokinetic properties, together with reducedtoxicity. It is expected that with the aforementioned advantages, theprodrug compounds of the invention will be efficient alternative noveldrugs for inflammatory-related diseases and disorders.

[0095] The first cleavage at position sn-2 of the phospholipid, mayfurther facilitate the following cleavage necessary for releasing theactive NSAID from the bridging group. This second cleavage may beenzymatically or non-enzymatically executed. Candidate enzymes forperforming the second cleavage may include an amidase, esterase or anyother suitable enzyme functionally available at the diseased site.

[0096] Alternatively, the release of an active anti-inflammatory drugfrom the D-X bond may be initiated by any cleavage at position sn-2 ofthe phospholipid that leads to release of an active drug. Moreover,under some circumstances the active drug released may be different fromthe original parent drug molecule. This includes drug derivativeswherein a chemical group(s) has been removed from or added to the Dstructure. These cases are also included within the concept of theinvention provided that the resulted drug derivative preserves itstherapeutic capability. Preferably the cleavage process of the moleculeof the invention is initiated specifically at the diseased cells, thusgenerating a highly specific and highly effective drug released at thedesired target site.

[0097] Irrespective of the exact mechanism of action, it is evident thatthe novel compounds of the invention have an enhanced therapeuticprofile. Furthermore, they are more effective than their correspondingparent drugs in at least two aspects: (i) increased specificity, and(ii) decreased side effects. The compounds of the invention may enableextending the usefulness of NSAIDs to conditions that do not respond tolower doses of the drug as well as reducing undesirable side effects bypreferential releasing of the active drug at the diseased site.

[0098] In accordance with another aspect of the invention, there areprovided pharmaceutical compositions comprising, as an activeingredient, a compound of the general formula I wherein Z of thebridging group having 3 to 15 carbon atoms, and a pharmaceuticallyacceptable diluent or carrier as are known in the art.

[0099] The pharmaceutical compositions may be in a liquid, aerosol orsolid dosage form, and may be formulated into any suitable formulationincluding, but not limited to, solutions, suspensions, micelles,emulsions, microemulsions, aerosols, ointments, gels, suppositories,capsules, tablets, and the like, as will be required for the appropriateroute of administration.

[0100] Compounds of the invention are useful in the treatment ofdiseases and disorders related to an inflammatory condition. Thus, inyet another aspect, the present invention provides a method for treatingsuch an inflammatory-related disease or disorder, comprisingadministering to an individual in need thereof a therapeuticallyeffective amount of a compound of the general formula I or apharmaceutical composition in accordance with the invention. The term“therapeutically effective amount” used in the specification refers tothe amount of a given prodrug compound according to the invention whichantagonizes or inhibits activities associated with inflammatoryprocesses, hence providing either a subjective relief of a symptom(s) oran objectively identifiable improvement as noted by the clinician orother qualified observer.

[0101] In particular, the present invention provides a method fortreating a disease or disorder related to inflammatory conditionincluding, but not being limited to, autoimmune diseases such asarthritis, rheumatoid arthritis, multiple sclerosis, systemic lupuserythematosus, other diseases such as asthma, psoriasis, inflammatorybowel syndrome, neurological degenerative diseases such as Alzheimer'sdisease, Parkinson's disease, Huntington's disease, vascular dementia,and other pathological conditions such as epilepsy, migraines, strokeand trauma.

[0102] Any suitable route of administration is encompassed by theinvention including, but not being limited to, oral, intravenous,intramuscular, subcutaneous, inhalation, intranasal, topical, rectal orother known routes. In preferred embodiments, the pharmaceuticalcompositions of the invention are orally or nasally administered.

[0103] The dose ranges for the administration of the compositions of theinvention are those large enough to produce the desired protectiveeffect. The dosing range of the prodrug varies with the specific drugused, the treated inflammatory condition or neurological disorder, theroute of administration and the potency of the particular prodrugmolecule in releasing the drug at the specific target site. The dosageadministered will be dependent upon the age, sex, health, weight of therecipient, concurrent treatment, if any, frequency of treatment and thenature of the effect desired. Dosage regimen and means of administrationwill be determined by the attending physician or other person skilled inthe art.

[0104] In still another aspect, the present invention provides a methodfor synthesizing compounds of the above-defined formula I by followingthe steps of the detailed scheme of synthesis described hereinbelow. Aparticular synthesis scheme is depicted in Scheme I and exemplified inExamples 1 to 5. Generally speaking the synthesis process involves thefollowing steps:

[0105] Step 1: protection of the functional moiety X on the bridginggroup. The purpose of this step is to prevent chemical reactions of Xduring the coupling of the bridging group to the lyso-lecithin molecule,so that linking of the bridging group to the lipid is exclusivelymediated through the carboxyl group of the linker HO(O)C-Z-X.

[0106] Any blocking group that reacts with the functional moiety X tomask its reactive function and is readily removable after coupling, maybe employed. Reagents suitable for use as protecting groups are wellknown to those skilled in the art and include, but are not limited to,the following: benzyl chloromate, benzyloxycarbonate (for NH₂ or NHprotection), benzyloxymethyl chloride, dihydropyran (for OH protection),diphenylcarbinol, trimethylacetamidocarbinol (for SH protection) andmethoxymethyl chloride (for COOH protection).

[0107] Appropriate blocking reagents and protocols for their usage aredescribed in Protecting Groups by Kocienski, P. (Thieme foundation oforganic chemistry series, 1994) and in Protective Groups in OrganicSynthesis by Greene, T. and Wuts, P. (John Wiley & Sons, Inc. 1991), theteachings of which are incorporated herein by reference.

[0108] Step 2: Preparation of an anhydride of the protected bridginggroup. The formation of the anhydride is performed by employing areagent which removes one molecule of water from two protected bridginggroups. This reaction is preferably performed under inert atmosphere. Acommonly used reagent for this reaction is, for example,dicyclohexylcarbodiimide (DCC).

[0109] Step 3: Coupling of the protected bridging group to alyso-lecithin. This step is carried out by acylating the appropriatephospholipid at position sn-2 to yield1-acyl-2-acyl(X-protected)-sn-glycero-3 phospholipid. The anhydride ofthe protected bridging group and the corresponding lyso-lecithin aredissolved in organic solvent, for example chloroform or methylenechloride, in the presence of a catalyst, for example a tret-amine suchas dimethylaminopyridine (DMAP).

[0110] Step 4: Removal of the blocking group from the functional groupX. Protocols for removal of the blocking groups used in step 1 forprotecting the functional group X, are disclosed in Protecting Groups byKocienski, P. (Thieme foundation of organic chemistry series, 1994) andin Protective Groups in Organic Synthesis by Greene, T. and Wuts, P.(John Wiley & Sons, Inc. 1991). In a particular procedure, theprotecting group is removed by hydrogenation in the presence of Pd/C.

[0111] Step 5: Coupling a nonsteroidal anti-inflammatory drug to thelipid moiety. Coupling of the corresponding drug to the functional groupX of the bridging group is the last stage in the protocol for thesynthesis of the compounds in accordance with the invention. Thisreaction is conducted in an organic solvent in the presence of reagentsthat enable a condensation reaction where water molecules are removed.Such commonly used reagents are, for example, the combination oftriphenylphosphine and aldrithiol-2.

[0112] Contrary to other known procedures for the synthesis of lipidderivatives of drugs, the present protocol is unique in designating thedrug conjugation step as the final one. It is important that the drug isadded at the last step in order to prevent possible modifications anddeterioration to its structure. Thus the process disclosed in thepresent invention is advantageous in terms of higher yield of thedesired reaction product and much reduced levels of side products.

[0113] In a preferred embodiment, the reacting functional groups thatform the X-D bond are —C(O)OH and NH₂ or NH groups, yielding a peptidebond. The carboxyl or the amino groups may be provided by either group Xon the bridging group, or as an available functional group on the drugmolecule. According to that preferred embodiment, when the reactinggroup of the drug is a carboxyl, it is reacted with an amine on thebridging group, and vice versa, when the reacting group of the drug isan amine, its reacting counterpart on the bridging group is a carboxyl.

EXAMPLES

[0114] A particular scheme for the synthesis of compounds of theinvention is outlined in Scheme I. This scheme is exemplified below, inExamples 1 to 5, by the detailed description of the synthesis ofspecific lipid derivatives of diclofenac (o-(2,6-dichloroanilino)phenylacetic acid), indomethacin,(1-(p-chlorobenzoyl)-5-methoxy-2-methyl-3-indolylacetic acid), ibuprofen(2-(4-isobutylphenyl)propionic acid), naproxen(d-2-(6-methoxy-2-naphthyl)propionic acid) and6-methoxy-2-naphthylacetic acid conjugated to phosphatidylcholine. Thissynthesis is a six-stage process: The first stage is protection of thefunctional group on the linker, in this case the amino group of an aminoacid. The second stage is preparation of anhydride of this protectedamino acid. The third stage is the formation of lipid derivativecomprising the protected amino acid and a lyso-lecithin. The removal ofthe protecting group to yield the amino acid lipid conjugate is carriedout in the fourth and fifth stages. Linking of the corresponding drug,in these particular examples, diclofenac, indomethacin, ibuprofen,naproxen or 6-methoxy-2-naphthylacetic acid to1-acyl-2-(n-amino)acyl-sn-glycero-3-phosphatidylcholine is realized inthe last stage.

[0115] Wherein HOOC-R_(d) in the synthesis scheme is a non-steroidalanti-inflammatory drug. For example, HOOC-R_(d) may be selected from:

Example 1 Preparation of Lipid Derivatives of Diclofenac (DP-DFC)

[0116] Stage 1. Protection of the Amino Group of Amino Acid (Preparationof Z-Amino Acid).

[0117] To a mixture of 0.1 mol corresponding amino acid (aminopropanoicacid, aminobutanoic acid, aminovaleric acid, aminohexanoic acid oraminooctanoic acid) in ethanol (25 ml) in round-bottom flask (250 ml)equipped with a magnetic stirrer and dropped funnel, a solution of NaOH(8.8 g., 0.22 mol) in 100 ml water is added and the mixture is stirredby magnetic stirrer until fully dissolved. The obtained solution iscooled to 0° C. in an ice-water bath, and benzyl chloroformate (27.4 g,0.15 mol) is added drop wise over 30 min. The reaction mixture isstirred for 3 hours at 0° C. Subsequently, about 100 ml water is addedto the reaction solution and the mixture is poured into separatedfunnel. The solution is washed with diethyl ether (3×50 ml). The waterfraction is separated and acidified with HCl (3N) to pH=1 while coolingin an ice-water bath. If a precipitate is formed, it is filtered, washedwith water and dissolved in 100 ml chloroform. The chloroform solutionis dried with sodium sulfate for two hours. Then the sodium sulfate isseparated from the chloroform solution by filtration and the solvent isevaporated in evaporator under vacuum. The residue is washed withhexane, and dried overnight in vacuum over phosphorus pentoxide (P₂O₅).

[0118] If the precipitate is not formed, or in order to maximize theproduct yield, the acidified aqueous fraction is washed with chloroform(2×50 ml). The chloroform extracts are combined and washed with water(50 ml). The following operations with this solution are the same as forthe above-described chloroform solution of the precipitate, namely,drying with sodium sulfate for two hours, then separating the sodiumsulfate from the chloroform solution by filtration and evaporating thesolvent in evaporator under vacuum. The residue is then washed withhexane, and dried overnight in vacuum over phosphorus pentoxide (P₂O₅).

[0119] All products were analyzed on TLC as follows:

[0120] TLC analysis. Silica gel 60 on aluminum sheet. Eluent ischloroform-methanol (4:1 v/v). Indicator is a spray of the composition:4-methoxybenzaldehyde (10 ml), absolute ethanol (200 ml), 98% sulfuricacid (10 ml) and glacial acetic acid (2 ml). The chromatogram is sprayedwith the indicator and then charred using hot air at 150-180° C.

[0121] The following are the intermediate products resulted at the endof stage 1 of the synthesis procedure:

[0122] 3-(Carbobenzyloxyamino)propanoic acid.C₆H₅—CH₂—O—C(O)—NH—CH₂—CH₂—COOH.

[0123] White solid. Yield 60%. TLC analysis: One spot R_(f) 0.7.

[0124]¹H NMR (CD₃OD), δ(ppm): 2.46-2.52 (t, 2H), 3.29-3.39 (t, 2H) 5.06(s, 2H), 7.27-7.32 (m, 5H).

[0125] 4-(Carbobenzyloxyamino)butanoic acid.C₆H₅CH₂—O—C(O)—NH—(CH₂)₃—COOH.

[0126] White solid. Yield 60%. TLC analysis: One spot. R_(f) 0.7.

[0127]¹H NMR (CD₃OD), δ (ppm): 1.71-1.82 (m, 2H), 2.28-2.34 (t, 2H),3.10-3.17 (t, 2H), 5.06 (s, 2H), 7.26-7.34 (m, 5H).

[0128] 5-(Carbobenzyloxyamino)valeric acid.C₆H₅CH₂—O—C(O)—NH—(CH₂)₄—COOH.

[0129] White solid. Yield 60%. TLC analysis: One spot. R_(f) 0.7.

[0130]¹H NMR (CD₃OD), δ (ppm): 1.45-1.50 (m, 2H), 1.56-1.62 (m, 2H),2.25-2.31 (t, 2H), 3.08-3.13 (t, 2H), 5.05 (s, 2H), 7.26-7.34 (m, 5H).

[0131] 6-(Carbobenzyloxyamino)hexanoic acid.C₆H₅CH₂—O—C(O)—NH—(CH₂)₅—COOH.

[0132] White solid. Yield 50%. m.p. 54-56° C. TLC analysis: One spot.R_(f) 0.7.

[0133]¹H NMR (CD₃OD), δ (ppm): 1.30-1.63 (several m, 6H), 2.24-2.30 (t,2H), 3.07-3.13 (t, 2H), 5.05 (s, 2H), 7.29-7.34 (m, 5H).

[0134] 8-(Carbobenzyloxyamino)octanoic acid.C₆H₅CH₂—O—C(O)—NH—(CH₂)₇—COOH.

[0135] White solid. Yield 50%. TLC analysis: One spot. R_(f) 0.7.

[0136]¹H NMR (CD₃OD), δ (ppm): 1.32 (broad s, 6H), 1.47-1.50 (m, 2H),1.53-1.59 (m, 2H), 2.23-2.29 (t, 2H), 3.06-3.12 (t, 2H), 5.05 (s, 2H),7.29-7.34 (s, 5H).

[0137] 12-(Carbobenzyloxyamino)dodecanoic acid.C₆H₅CH₂—O—C(O)—NH—(CH₂)₁₁—COOH.

[0138] White solid. Yield 50%. TLC analysis: One spot. R_(f) 0.7.

[0139]¹H NMR (CD₃OD), δ (ppm): 1.30 (broad s, 14H), 1.48-1.51 (m, 2H),1.54-1.59 (m, 2H), 2.25-2.29 (t, 2H), 3.06-3.13 (t, 2H), 5.05 (s, 2H),7.29-7.34 (s, 5H).

[0140] Stage 2. Synthesis of Z-Amino Acid Anhydride.

[0141] The solution of corresponding Z-amino acid produced at stage 1(0.05 mol) in freshly distilled dichloromethane (25 ml) is introduced,under an inert atmosphere of argon, into double-neck round-bottomequipped with magnetic stirrer and dropped funnel. A solution ofdicyclohexylcarbodiimide (0.0325 mol) in 25 ml of freshly distilleddichloromethane, also under argon, is added drop wise, with stirring, tothe solution of Z-amino acid. After 20 min of stirring, the obtainedprecipitate of urea is filtered and the solution evaporated undervacuum. The crude residue is washed with hexane (2×20 ml) and then driedin vacuum.

[0142] TLC analysis: The same procedure is used for TLC analysis of theanhydrides of all Z-amino-acids. Silica gel 60 on aluminum sheet. Fluentis the mixture of chloroform with acetone (8:2, v/v). For indication,ninhydrine spray is used on the chromatogram followed by charring withhot air (100° C.).

[0143] Anhydride of Z-(3-amino)propanoic acid.

[0144] White solid. Yield is 70%. TLC analysis: One spot R_(f) 0.8.

[0145] Chemical analysis. C₂₂H₂₄N₂O₇. Calculated: C 61.68%, H 5.60%, N6.54%. Found: C 61.20%, H 5.52%, N 6.50%.

[0146] Anhydride of Z-(4-amino)butanoic acid.

[0147] White solid. Yield is 70%. TLC analysis: One spot. R_(f) 0.8.

[0148] Chemical analysis. C₂₄H₂₈N₂O₇. Calculated: C 63.16%, H 6.14%, N6.14%. Found: C 62.77%, H 6.36%, N 5.88%.

[0149] Anhydride of Z-(5-amino)valeric acid.

[0150] White solid. Yield is 70%. TLC analysis: One spot. R_(f) 0.8.

[0151] Chemical analysis. C₂₆H₃₂N₂O₇. Calculated: C 64.46%, H 6.61%, N5.78%. Found: C 64.09%, H 6.86%, N 5.49%.

[0152] Anhydride of Z-(6-amino)hexanoic acid.

[0153] White solid. Yield is 70%. TLC analysis: One spot. R_(f) 0.8.

[0154] Chemical analysis: C₂₈H₃₆N₂O₇. Calculated: C 64.46%, H 6.61%, N5.79%. Found: C 64.39%, H 6.85%, N 5.52%.

[0155] Anhydride of Z-(8-amino)octanoic acid.

[0156] White solid. Yield is 75%. TLC analysis: One spot. R_(f) 0.85.

[0157] Chemical analysis: C₃₂H₄₄N₂O₇. Calculated: C 67.60%, H 7.75%, N4.93%. Found: C 67.44%, H 7.79%, N 4.72%.

[0158] Anhydride of z-(12-amino)dodecanoic acid.

[0159] White solid. Yield is 70%. TLC analysis: One spot. R_(f) 0.85.

[0160] Chemical analysis: C₄₀H₆₀N₂O₇. Calculated: C 72.29%, H 9.04%, N4.21%. Found: C 72.02%, H 9.42%, N 4.12%.

[0161] Stage 3. Preparation of1-acyl-2-(Z-amino)acyl-sn-glycero-3-phosphotidylcholine.

[0162] The anhydride of the corresponding Z-amino acid, 0.01 moldissolved in 150 ml of freshly distilled chloroform, is introduced,under an inert atmosphere of argon, into a single-neck round-bottomflask (250 ml) equipped with a magnetic stirrer. To this solution 0.01mol (1.22 g) 4-(dimethylamino)pyridine (DMAP) in 25 ml chloroform isadded, followed by addition of a suspension of 0.0056 moleslyso-lecithin in 50 ml of chloroform. The reaction mixture is vigorouslystirred for 3-5 hours at room temperature. The lyso-lecithin dissolvesand reaction mixture becomes transparent after about 2 hours ofstirring. The reaction is monitored by TLC using silica gel 60 onaluminum sheet, the eluent is chloroform:methanol:water, 65:35:5, theindicator is a spray of the composition: 4-methoxybenzaldehyde (10 ml),absolute ethanol (200 ml), 98% sulfuric acid (10 ml) and glacial aceticacid (2 ml). The chromatogram is sprayed with the indicator followed bycharring with hot air at 150° C. The reaction is assumed to be completeand stopped when all the lyso-lecithin has disappeared. The reactionmixture is then transferred into a separating funnel and washed with asolution of 1% HCl (3×50 ml), then with saturated solution of sodiumbicarbonate (3×50 ml) and finally with water (3×50 ml). The obtainedproduct in the organic solution is dried over sodium sulfate and thenfiltered. The solvent is evaporated at 30° C. in vacuo and the residueis washed with hexane and left to dry overnight under vacuum. Theresulted molecule1-acyl-2-(Z-amino)acyl-sn-glycero-3-phosphatidylcholine is the mainproduct of the reaction.

[0163] The second product of the reaction is the Z-amino acid. In orderto increase the yield of this product, it is back-extracted fromreaction mixture as follows: The sodium bicarbonate aqueous fractionsare collected and combined and then acidified by 3 N HCl to pH 1. TheZ-amino acid is extracted by chloroform (2×50 ml). The chloroformextracts were combined, washed once with water and dried over sodiumsulfate for 30 min with stirring. The sodium sulfate is removed byfiltration, and the chloroform evaporated. Subsequently, the residue iswashed with hexane and dried over P₂O₅ in vacuo.

[0164] TLC analysis: Silica gel 60 on aluminum sheet. Eluent ischloroform/methanol/water (65:35:5, v/v). Indicator is a spray of thecomposition: 4-methoxybenzaldehyde (10 ml), absolute ethanol (200 ml),98% sulfuric acid (10 ml) and glacial acetic acid (2 ml). Thechromatogram is sprayed with the indicator and then charred using hotair at 100-150° C.

[0165]1-Stearoyl-2-[3′-(carbobenzyloxyamino)]propanoyl-sn-glycero-3-phosphotidylcholine.

[0166] White wax. Yield 70%. TLC analysis: One spot. R_(f) 0.55

[0167]¹H NMR (CDCl₃), δ (ppm): 0.83-0.89 (t, 3H), 1.23-1.27 (broad s,28H), 1.54 (m, 2H), 2.22-2.29 (t, 2H), 2.53-2.56 (m, 2H), 3.15 (s, 9H),3.41-3.44 (m, 2H), 3.60-3.63 (m, 2H), 3.85-3.96 (m, 2H), 4.13-4.25 (m,4H), 5.05 (s, 2H), 5.20 (m, 1H), 7.27-7.33 (m, 5H).

[0168]1-Stearoyl-2-[4′-(carbobenzyloxyamino)]butanoyl-sn-glycero-3-phosphotidylcholine.

[0169] White wax. Yield 70%. TLC analysis: One spot. R_(f) 0.55.

[0170]¹H NMR (CDCl₃), δ (ppm): 0.84-0.88 (t, 3H), 1.25 (broad s, 28H),1.52-1.55 (m, 2H), 1.72-1.80 (m, 2H), 2.23-2.32 (m, 4H), 3.07-3.14 (m,2H), 3.18 (s, 9H), 3.61-3.65 (m, 2H), 3.86-3.94 (m, 2H), 4.10-4.25 (m,4H), 5.06 (s, 2H), 5.22 (m, 1H), 7.26-7.33 (m, 5H).

[0171]1-Stearoyl-2-[5′-(carbobenzyloxyamino)]valeroyl-sn-glycero-3-phosphotidylcholine.

[0172] White wax. Yield 70%. TLC analysis: One spot. R_(f) 0.55.

[0173]¹H NMR (CDCl₃), δ (ppm): 0.84-0.89 (t, 3H), 1.26 (broad s, 28H),1.54-1.65 (m, 4H), 1.72-1.77 (m, 2H), 2.23-2.30 (m, 4H), 3.07-3.12 (m,2H), 3.16 (s, 9H), 3.61-3.65 (m, 2H), 3.86-3.94 (m, 2H), 4.10-4.25 (m,4H), 5.06 (s, 2H), 5.20 (m, 1H), 7.26-7.33 (m, 5H).

[0174]1-Stearoyl-2-[6′-(carbobenzyloxyamino)]hexanoyl-sn-glycero-3-phosphotidylcholine.

[0175] White wax. Yield 65%. TLC analysis: One spot. R_(f) 0.55.

[0176]¹H NMR (CDCl₃), δ (ppm): 0.84-0.89 (t, 3H), 1.24 (broad s, 28H),1.30-1.62 (several m, 8H), 2.22-2.30 (m, 4H), 3.06-3.12 (m, 2H), 3.15(s, 9H), 3.61-3.65 (m, 2H), 3.88-3.97 (m, 2H), 4.10-4.25 (m, 4H), 5.05(s, 2H), 5.20 (m, 1H), 7.25-7.32 (m, 5H).

[0177]1-Stearoyl-2-[8′-(carbobenzyloxyamino)]octanoyl-sn-glycero-3-phosphotidylcholine.

[0178] White wax. Yield 65%. TLC analysis: One spot. R_(f) 0.55.

[0179]¹H NMR (CDCl₃), δ (ppm): 0.84-0.89 (t, 3H), 1.25 (broad s, 28H),1.30-1.33 (m, 6H), 1.46-1.49 (m, 2H), 1.52-1.58 (m, 4H), 2.22-2.29 (m,4H), 3.05-3.10 (m, 2H), 3.17 (s, 9H), 3.61-3.65 (m, 2H), 3.85-3.96 (m,2H), 4.10-4.23 (m, 4H), 5.06 (s, 2H), 5.20 (m, 1H), 7.22-7.29 (m, 5H).

[0180]1-Stearoyl-2-[12′-(carbobenzyloxyamino)]dodecanoyl-sn-glycero-3-phosphotidylcholine.

[0181] White solid. Yield 60%. TLC analysis: One spot. R_(f) 0.53.

[0182]¹H NMR (CDCl₃), δ (ppm): 0.84-0.88 (t, 3H), 1.23 (broad s, 28H),1.30 (broad s, 14H), 1.45-1.50 (m, 4H), 1.53-1.59 (m, 2H), 2.23-2.50 (m,4H), 3.06-3.12 (m, 2H), 3.17 (s, 9H), 3.62-3.67 (m, 2H), 3.84-3.94 (m,2H), 4.10-4.25 (m, 4H), 5.05 (s, 2H), 5.20 (m, 1H), 7.23-7.30 (m, 5H).

[0183] Stage 4. Removal of the Protecting Benzyloxycarbonyl Group.

[0184] The obtained1-stearoyl-2-(carbobenzyloxyamino)acyl-3-phosphotidylcholine (0.0025mol) is dissolved in a mixture of 100 ml methanol and 5 ml acetic acid.The solution is introduced into round bottom double neck flask (200 ml)equipped with a magnetic stirrer, under an atmosphere of argon. Pd/C(0.5 g) is added to the solution and hydrogen is blown through thereaction mixture for 4 hours. The reaction proceeding is monitored byTLC analysis under the following conditions: silica gel 60 on aluminumsheet, eluent is the mixture of chloroform/methanol/water (65:35:5,v/v), indicator is a spray of the composition: p-methoxybenzaldehyde (10ml), absolute ethanol (200 ml), 98% sulfuric acid (10 ml) and glacialacetic acid (2 ml). The chromatogram is sprayed with the indicator andthen charred using hot air at 100-150° C.

[0185] The reaction assumed to be complete and hydrogenation is stoppedafter all corresponding1-stearoyl-2-carbobenzyloxyaminoacyl-sn-glycero-phosphatidylcholine hasdisappeared. The reaction mixture is then filtered through celete toremove the Pd/C, evaporated at 30° C., under vacuum. The crude residueis washed with ether (3×30 ml) and dried in vacuo overnight. Conditionsof the TLC analysis are the same as indicated above.

[0186] 1-Stearoyl-2-(3-amino)propanoyl-sn-glycero-3-phosphatidylcholine,acetic acid.

[0187] White wax. Yield 70%. TLC analysis: One spot. R_(f) 0.2.

[0188]¹H NMR (CD₃OD), δ (ppm): 0.87-0.92 (t, 3H), 1.28 (broad s, 28H),1.58-1.61 (m, 2H), 1.92 (s, 3H), 2.30-2.38 (t, 2H), 2.71-2.77 (t, 2H),3.17-3.19 (m, 2H), 3.22 (s, 9H), 3.62-3.65 (m, 2H), 3.87-4.48 (severalm, 6H), 5.24 (m, 1H).

[0189]³¹P NMR (CD₃OD), δ (ppm): 0.01 (s).

[0190] Chemical analysis: C₂₉H₅₉N₂O₈P.CH₃COOH. Calculated: C 56.88%, H9.63%, N 4.28%, P 4.74%. Found: C 57.01%, H 10.11%, N 4.18%, P 4.52%.

[0191] 1-Stearoyl-2-(4-amino)butanoyl-sn-glycero-3-phosphatidylcholine,acetic acid.

[0192] White wax. Yield 70%. TLC analysis: One spot. R_(f) 0.2.

[0193]¹H NMR (CD₃OD), δ (ppm): 0.86-0.92 (t, 3H), 1.28 (broad s, 28H),1.56-1.60 (m, 2H), 1.94 (s, 3H), 1.96-1.99 (m, 2H), 2.29-2.35 (t, 2H),2.46-2.52 (m, 2H), 2.95-3.02 (t, 2H), 3.22 (s, 9H), 3.62-3.66 (m, 2H),3.87-4.46 (several m, 6H), 5.21-5.22 (m, 1H).

[0194]³¹P NMR (CD₃OD), δ (ppm): 0.01 (s).

[0195] Chemical analysis: C₃₀H₆₁N₂O₈P.CH₃COOH. Calculated: C 57.48%, H9.73%, N 4.19%, P 4.64%. Found: C 57.12%, H 9.55%, N 4.22%, P 4.38%.

[0196] 1-Stearoyl-2-(5-amino)valeroyl-sn-glycero-3-phosphatidylcholine,acetic acid

[0197] White wax. Yield 70%. TLC analysis: One spot. R_(f) 0.2.

[0198]¹H NMR (CD₃OD), δ (ppm): 0.87-0.92 (t, 3H), 1.27 (broad s, 28H),1.55-1.61 (m, 2H), 1.92 (s, 3H), 1.96-2.10 (m, 2H), 2.29-2.35 (t, 2H),2.46-2.52 (m, 2H), 2.95-3.02 (t, 2H), 3.22 (s, 9H), 3.62-3.66 (m, 2H),3.85-4.46 (several m, 6H), 5.19-5.21 (m, 1H).

[0199]³¹P NMR (CD₃OD), δ (ppm): 0.01 (s).

[0200] Chemical analysis: C₃₁H₆₃N₂O₈P.CH₃COOH. Calculated: C 58.06%, H9.82%, N 4.05%, P 4.54%. Found: C 57.90%, H 10.01%, N 4.20%, P 4.48%.

[0201] 1-Stearoyl-2-(6-amino)hexanoyl-sn-glycero-3-phosphatidylcholine,acetic acid.

[0202] White wax. Yield 65%. TLC analysis: One spot. R_(f) 0.2.

[0203]¹H NMR (CD₃OD), δ (ppm): 0.86-0.92 (t, 3H), 1.28 (broad s, 28H),1.32-1.50 (m, 2H), 1.56-1.72 (broad m, 6H), 1.91 (s, 3H), 2.28-2.40(broad m, 4H), 2.88-2.94 (t, 2H), 3.22 (s, 9H), 3.61-3.66 (m, 2H),3.95-4.08 (m, 2H), 4.10-4.38 (several m, 4H), 5.24 (m, 1H).

[0204]³¹P NMR (CD₃OD), δ (ppm): 0.01 (s).

[0205] Chemical analysis: C₃₂H₆₅N₂O₈P.CH₃COOH. Calculated: C 58.62%, H9.91%, N 4.02%, P 4.45%. Found: C 58.19%, H 10.11%, N 4.12%, P 4.58%.

[0206] 1-Stearoyl-2-(8-amino)octanoyl-sn-glycero-3-phosphatidylcholine,acetic acid.

[0207] White wax. Yield 65%. TLC analysis: One spot. R_(f) 0.2.

[0208]¹H NMR (CD₃OD), δ (ppm): 0.86-0.92 (t, 3H), 1.28 (broad s, 28H),1.37 (s, 6H), 1.59-1.67 (broad m, 6H), 1.93 (s, 3H), 2.28-2.39 (broad m,4H), 2.86-2.92 (t, 2H), 3.22 (s, 9H), 3.62-3.66 (m, 2H), 3.97-4.03 (m,2H), 4.13-4.39 (several m, 4H), 5.22-5.23 (m, 1H).

[0209]³¹P NMR (CD₃OD), δ (ppm): 0.01 (s).

[0210] Chemical analysis: C₃₄H₆₉N₂O₈P.CH₃COOH. Calculated: C 59.67%, H10.08%, N 3.87%, P 4.28%. Found: C 59.22%, H 10.21%, N 3.99%, P 4.08%.

[0211]1-Stearoyl-2-(12-amino)dodecanoyl-sn-glycero-3-phosphatidylcholine,acetic acid. White solid. Yield 60%. TLC analysis: One spot. R_(f) 0.2.

[0212]¹H NMR (CD₃OD), δ (ppm): 0.85-0.90 (t, 3H), 1.28 (s, 28H), 1.36(broad s, 14H), 1.56-1.68 (m, 6H), 1.94 (s, 3H), 2.29-2.39 (m, 4H),2.85-2.90 (m, 2H), 3.20 (s, 9H), 3.60-3.66 (m, 2H), 3.95-4.03 (m, 2H),4.12-4.37 (several m, 4H), 5.21-5.23 (m, 1H).

[0213]³¹P NMR (CD₃OD), δ (ppm): 0.01 (s).

[0214] Chemical analysis: C₄₀H₈₁N₂O₁₀P.CH₃COOH. Calculated: C 61.54%, H10.38%, N 3.59%, P 3.97%. Found: C 61.36%, H 10.69%, N 3.41%, P 3.85%.

[0215] Stage 5. Preparation of Free1-acyl-2-[n(amino)acyl-sn-glycero-3-phosphatidylcholine

[0216] The solution of acetic acid and the corresponding1-acyl-2-[n-aminoacyl-sn-glycero-3-phosphatidylcholine complex (1.36mmol) in methylene chloride (30 ml) is introduced into a single neckround bottom flask (150 ml) equipped with a magnetic stirrer.Triethylamine (0.3 ml, 3 mmol) is added to this solution. The resultingreaction mixture is stirred at room temperature for 30 min. During thisprocedure the free 2-amino-acyl lipid is formed. The reaction solutionwith the obtained free 2-amino-acyl lipid is used for the followingsynthesis without any additional processing.

[0217] Stage 6. Preparation of1-acyl-2-n(arylacetamido)acyl-sn-glycero-3-phosphatidylcholine

[0218] A mixture of the corresponding drug, in this case diclofenac[o-[(2,6-dichlorophenyl)amino]phenyl]acetic acid] (1.36 mmol),triphenylphosphine (0.72 g., 2.75 mmol) and aldritiol-2 (0.6 g., 2.75mmol) is introduced, under an inert atmosphere of argon, into a reactionflask containing the solution of free 2-amino-acyl lipid in methylenechloride (see stage 5). The reaction solution immediately becomes yellowand is left stirring for one hour at room temperature in an atmosphereof argon. The solvent is then removed by evaporation and the product isextracted and purified by a flash column chromatography by varying thecomposition of the mobile phase. For extracting and purifying 1 g. ofsolid reaction mixture, 35 g. of silica gel 60 (230-400 mesh ASTM) in aglass column (40×2 cm) with a pressure of about 1.5 atm., was used. Thefirst fraction of eluent is 150 ml of a mixture of chloroform withmethanol (65:35, v/v). The second fraction of eluent is 250 ml of amixture of chloroform, methanol and water (65:35:5, v/v). In the firstfraction the main impurities are removed, and in the second fraction thefine purification of the final product, i.e. the phospholipid-drugconjugate, is realized.

[0219] Lipid Derivatives of Diclofenac (DP-DFC)

[0220] All the synthesized products mentioned below are pale yellowsolids that when analyzed by TLC are displayed in one bright red spot,R_(f) is 0.3. The TLC analysis conditions are as follows: Silica gel 60on aluminum sheet. Eluent is chloroform:methanol:water (65:35:5, v/v).Indicator is a spray of the composition: 4-methoxybenzaldehyde (10 ml),absolute ethanol (200 ml), 98% sulfuric acid (10 ml) and glacial aceticacid (2 ml). The chromatogram is sprayed with the indicator and thencharred at 100° C.

[0221]1-Stearoyl-2-{3-[2′-(2″,6″-dichloroanilino)phenylacetamido]propanoyl}-sn-glycero-3-phosphatidylcholine.

[0222] Yield 75%.

[0223]¹H NMR (CD₃OD), δ (ppm): 0.84-0.89 (t, 3H), 1.26 (broad s, 28H),1.54-1.59 (m, 2H), 2.27-2.33 (t, 2H), 2.56-2.61 (t, 2H), 3.18 (s, 9H),3.44-3.49 (t, 2H), 3.58-3.61 (m, 2H), 3.68 (s, 2H), 4.02-4.05 (m, 2H),4.19-4.26 (m,3H), 4.36-4.38 (m, 1H), 5.21-5.25 (m. 1H), 6.36-6.40 (d,1H), 6.84-6.90 (t, 1H), 7.02-7.09 (m,2H), 7.20-7.24 (d. 1H), 7.37-7.41(d, 2H).

[0224]³¹P NMR (CD₃OD), δ (ppm): −0.84 (s).

[0225] Chemical analysis: C₄₃H₆₈N₃O₉PCl₂.2H₂O. Calculated: C 56.83%, H7.93%, N 4.62%, P 3.41%. Found: C 57.01%, H 7.59/o, N 4.27%, P 3.39%.

[0226]1-Stearoyl-2-{4-[2′-(2″,6″-dichloroanilino)phenylacetamido]butanoyl}-sn-glycero-3-phosphatidylcholine.

[0227] Yield 75%.

[0228]¹H NMR (CD₃OD), δ (ppm): 0.86-0.90 (t, 3H), 1.25 (broad s, 28H),1.53-1.57 (m, 2H), 1.80-1.84 (m, 2H), 2.27-2.31 (t, 2H), 2.36-2.41 (t,2H), 3.17 (s, 9H), 3.19-3.25 (m, 2H), 3.57-3.60 (m, 2H), 3.66 (s, 2H),4.00-4.03 (m, 2H), 4.17-4.25 (m, 3H), 4.37-4.41 (m, 1H), 5.21-5.25 (m,1H), 6.37-6.40 (d, 1H), 6.85-6.89 (t, 1H), 7.01-7.07 (m, 2H), 7.19-7.22(d, 1H), 7.37-7.40 (d, 2H).

[0229]³¹P NMR (CD₃OD), δ (ppm): −0.88 (s).

[0230] MS: C₄₄H₇₀N₃O₉PCl₂, Found m/e: 886.9 (FAB) (main pick).

[0231] Chemical analysis: C₄₄H₇₀N₃O₉PCl₂.2H₂O. Calculated: C 57.27%, H8.03%, N 4.56%, P 3.36%. Found: C 57.21%, H 8.11%, N 4.61%, P 3.32%.

[0232]1-Stearoyl-2-[5-[2′-(2″,6″-dichloroanilino)phenylacetamido]valeroyl]-sn-glycero-3-phosphatidylcholine.

[0233] Yield 75%.

[0234]¹H NMR (CD₃OD), δ (ppm): 0.83-0.89 (t, 3H), 1.24 (broad s, 28H),1.54-1.64 (broad m, 6H), 2.26-2.34 (m, 4H), 3.18-3.23 (m, 11H),3.58-3.62 (m, 2H), 3.66 (s, 2H), 4.00-4.03 (m, 2H), 4.16-4.25 (m, 3H),4.36-4.38 (m, 1H), 5.21-5.25 (m, 1H), 6.36-6.40 (d, 1H), 6.87-6.89 (t,1H), 7.02-7.08 (m, 2H), 7.19-7.22 (d, 1H), 7.37-7.40 (d, 2H).

[0235]³¹P NMR (CD₃OD), δ (ppm): −0.92 (s).

[0236] Chemical analysis: C₄₅H₇₂N₃O₉PCl₂. Calculated: C 60.00%, H 8.00%,N 4.66%, P 3.44%, Cl 7.88%. Found: C 59.64%, H 8.28%, N 4.69%, P 3.54%,Cl 7.66%.

[0237]1-Stearoyl-2-{6-[2′-(2″,6″-dichloroanilino)phenylacetamido]hexanoy}-sn-glycero-3-phosphatidylcholine.

[0238] Yield 80%.

[0239]¹H NMR (CD₃OD), δ (ppm): 0.85-0.90 (t, 3H), 1.26-1.35 (broad s,30H), 1.50-1.61 (m, 6H), 2.26-2.34 (m, 4H), 3.16-3.22 (m, 11H),3.59-3.66 (m, 4H), 4.00-4.02 (m, 2H), 4.19-4.26 (several m, 3H),4.38-4.40 (m, 1H), 5.21-5.25 (m, 1H), 6.37-6.40 (d, 1H), 6.83-6.90 (t1H), 6.99-7.07 (several m, 2H), 7.18-7.22 (d, 1H), 7.37-7.41 (d, 2H).

[0240]³¹P NMR (CD₃OD), δ (ppm): −0.92 (s).

[0241] Chemical analysis: C₄₆H₇₄N₃O₉PCl₂.2H₂O. Calculated: C 58.10%, H8.21%, N 4.42%, P 3.26%. Found: C 58.31%, H 8.36%, N 4.11%, P 3.30%.

[0242]1-Stearoyl-2-{8-[2′-(2″,6″-dichloroanilino)phenylacetamido]octanoyl}-sn-glycero-3-phosphatidylcholine.

[0243] Yield 80%.

[0244]¹H NMR (CD₃OD), δ (ppm): 0.85-0.91 (t, 3H), 1.26-1.30 (broad s,34H), 1.51-1.60 (m, 6H), 2.26-2.34 (m, 4H), 3.16-3.22 (m, 11H),3.60-3.65 (m, 4H), 3.96-4.02 (m, 2H), 4.16-4.27 (several m, 3H),4.38-4.40 (m, 1H), 5.19-5.24 (m, 1H), 6.36-6.40 (d, 1H), 6.83-6.90 (t,1H), 6.99-7.09 (several m, 2H), 7.18-7.22 (d, 1H), 7.37-7.41 (d, 2H).

[0245]³¹P NMR (CD₃OD), δ (ppm): −0.91 (s).

[0246] Chemical analysis: C₄₈H₇₈N₃O₉PCl₂.2H₂O. Calculated: C 58.90%, H8.380/%, N 4.29%, P 3.16%. Found: C 59.19%, H 8.39%, N 4.19%, P 3.20%.

[0247]1-Stearoyl-2-{12-[2′-(2″,6″-dichloroanilino)phenylacetamido]dodecanoyl}-sn-glycero-3-phosphatidylcholine.

[0248] Yield 70%.

[0249]¹H NMR (CD₃OD), δ (ppm): 0.87-0.92 (t, 3H), 1.28 (broad s, 42H),1.49-1.62 (broad m, 6H), 2.28-2.36 (m, 4H), 3.16-3.22 (m+s, 11H),3.62-3.66 (m+s, 4H), 3.98-4.03 (t, 2H), 4.18-4.29 (m, 3H), 4.41-4.42 (d,1H), 5.20 (broad m, 1H), 6.37-6.41 (d, 1H), 6.87-6.90 (t, 1H), 7.02-7.10(m, 2H), 7.19-7.22 (d, 1H), 7.39-7.42 (d, 2H).

[0250]³¹P NMR (CD₃OD), δ (ppm): −1.71 (s).

[0251] Chemical analysis: C₅₂H₈₆N₃O₉PCl₂.H₂O. Calculated: C 61.42%, H8.66%, N 4.13%, P 3.05%. Found: C 61.33%, H 9.07%, N 4.18%, P 2.85%.

Example 2 Preparation of Lipid Derivatives of Indomethacin (DP-Indo)

[0252] The procedure for the preparation of lipid derivatives ofindomethacin (1-(p-chlorobenzoyl)-5-methoxy-2-methyl-3-indolylaceticacid) is the same as the process outlined in Example 1, steps 1 to 6,except that in step 6 instead of diclofenac the drug included in thereaction mixture is indomethacin.

[0253] Lipid Derivatives of Indomethacin (DP-Indo)

[0254] The synthesized compounds were subjected to TLC analysis underthe following conditions: Silica gel 60 on aluminum sheet. Eluent ischloroform:methanol:water (65:35:5, v/v). Indicator is a spray of thecomposition: 4-methoxybenzaldehyde (10 ml), absolute ethanol (200 ml),98% sulfuric acid (10 ml) and glacial acetic acid (2 ml). Thechromatogram is sprayed with the indicator and then charred at 100° C.

[0255]1-Stearoyl-2-{3-[1-(p-chlorobenzoyl)-5-methoxy-2-methyl-3-indolylacetamido]propanoyl}-sn-glycero-3-phosphatidylcholine.

[0256] Pale yellow wax. Yield 80%.

[0257] TLC analysis: One spot. R_(f) is 0.35.

[0258]¹H NMR (CD₃OD), δ (ppm): 0.85-0.91 (t, 3H), 1.26-1.30 (broad s,28H), 1.52-1.55 (m, 2H), 2.25-2.31 (m, 5H), 2.54-2.60 (t, 2H), 3.18 (m,9H), 3.43-3.46 (t, 2H), 3.57-3.62 (m, 4H), 3.81 (s, 3H), 3.98-4.02 (m,2H), 4.13-4.25 (several m, 4H), 5.16 (m, 1H), 6.64-6.69 (d, 1H),6.92-6.95 (d, 1H), 7.00 (s, 1H), 7.54-7.58 (d, 2H), 7.67-7.72 (d, 2H).

[0259]³¹P NMR (CD₃OD), δ (ppm): −0.13 (s).

[0260] Chemical analysis: C₄₈H₇₃N₃O₁₁PCl.2H₂O. Calculated: C 59.41%, H7.94%, N 4.33%, P 3.20%, Cl 3.66%. Found: C 59.79%, H 7.96%, N 3.91%, P3.28%, Cl 3.60%.

[0261]1-Stearoyl-2-{4-[1-(p-chlorobenzoyl)-5-methoxy-2-methyl-3-indolylacetamido]butanoyl}-sn-glycero-3-phosphatidylcholine.

[0262] Pale yellow wax. Yield 80%.

[0263] TLC analysis: One spot. R_(f) is 0.35.

[0264]¹H NMR (CD₃OD), δ (ppm): 0.85-0.91 (t, 3H), 1.26 (broad s, 28H),1.52-1.55 (m, 2H), 1.77-1.83 (m, 2H), 2.24-2.39 (m, 7H), 3.18 (s, 9H),3.21-3.29 (m, 2H), 3.57-3.60 (m, 4H), 3.80-3.81 (s, 3H), 3.98-4.01 (m,2H), 4.15-4.36 (several m, 4H), 5.19-5.20 (m, 1H), 6.64-6.69 (d, 1H),6.92-6.96 (d, 1H), 7.00 (s, 1H), 7.54-7.57 (d, 2H), 7.67-7.72 (d, 2H).

[0265]³¹P NMR (CD₃OD) δ (ppm): −0.08 (s).

[0266] Chemical analysis: C₄₉H₇₅N₃O₁₁PCl.H₂O. Calculated: C 60.90%. H7.98%, N 4.27%, P 3.15%, Cl 3.67%. Found: C 60.82%, H 8.35%, N 4.27%, P3.10%, Cl 3.60%.

[0267]1-Stearoyl-{2-5-[1-(p-chlorobenzoyl)-5-methoxy-2-methyl-3-indolylacetamido]valeroyl}-sn-glycero-3-phosphatidylcholine.

[0268] Pale yellow wax. Yield 80%.

[0269] TLC analysis: One spot. R_(f) is 0.35.

[0270]¹H NMR (CD₃OD), δ (ppm): 0.85-0.91 (t, 3H), 1.26 (s, 28H),1.53-1.60 (m, 6H), 2.25-2.36 (m, 7H), 3.21 (s, 9H), 3.58-3.62 (m, 4H),3.87-3.80 (s, 3H), 3.98-4.01 (m, 2H), 4.15-4.24 (several m, 3H),4.35-4.37 (two d, 1H), 5.20-5.21 (m, 1H), 6.64-6.68 (d, 1H), 6.92-6.96(d, 1H), 7.00 (s, 1H), 7.54-7.57 (d, 2H), 7.67-7.72 (d, 2H)

[0271]³¹P NMR (CD₃OD), δ (ppm): −0.07(s).

[0272] Chemical analysis: C₅₀H₇₇N₃O₁₁PCl.2H₂O. Calculated: C 60.15%. H8.12%, N 4.21%, P 3.11%, Cl 3.56%. Found: C 60.39%, H 8.33%, N 4.08%, P3.05%, Cl 3.50%.

[0273]1-Stearoyl-2-[6-[1-(p-chlorobenzoyl)-5-methoxy-2-methyl-3-indolylacetamido]hexanoyl}-sn-glycero-3-phosphatidylcholine.

[0274] Pale yellow wax. Yield 80%.

[0275] TLC analysis: One spot. R_(f) is 0.38.

[0276]¹H NMR (CD₃OD), δ (ppm): 0.85-0.91 (t, 3H), 1.26 (broad s, 30H),1.48-1.62 (m, 6H), 2.26-2.32 (m, 7H), 3.15-3.19 (m, 11H), 3.59-3.62 (m,4H), 3.80 (s, 3H), 3.96-4.01 (t, 2H), 4.16-4.25 (m, 3H), 4.36-4.38 (twod, 1H), 5.20-5.21 (m, 1H), 6.64-6.69 (d, 1H), 6.91-6.95 (d, 1H), 7.01(s, 1H), 7.53-7.58 (d, 2H), 7.68-7.71 (d, 2H)

[0277]³¹P NMR (CD₃OD), δ (ppm): −0.07(s).

[0278] Chemical analysis: C₅₁H₇₉N₃O₁₁PCl.H₂O. Calculated: C 61.60%, H8.20%, N 4.22%, P 3.12%, Cl 3.57%. Found: C 60.08%, H 8.35%, N 4.27%, P3.22%, Cl 3.60%.

[0279]1-Stearoyl-2-{8-[1-(p-chlorobenzoyl)-5-methoxy-2-methyl-3-indolylacetamido]octanoyl}-sn-glycero-3-phosphatidylcholine.

[0280] Pale yellow wax. Yield 80%.

[0281] TLC analysis: One spot. R_(f) is 0.38.

[0282]¹H NMR (CD₃OD), δ (ppm): 0.85-0.91 (t, 3H), 1.26 (broad s, 34H),1.51-1.57 (m, 6H), 2.26-2.33 (m, 7H) 3.16-3.20 (m, 11H), 3.58-3.63 (m,4H), 3.80 (s, 3H), 3.96-4.02 (t, 2H), 4.16-4.26 (m, 3H), 4.39-4.41 (twod, 1H), 5.21 (m, 1H), 6.64-6.69 (d, 1H), 6.91-6.95 (d, 1H), 7.01 (s,1H), 7.53-7.58 (d, 2H), 7.68-7.71 (d, 2H)

[0283]³¹P NMR (CD₃OD), δ (ppm): −0.08(s).

[0284] Chemical analysis: C₅₃H₈₃N₃O₁₁PCl.2H₂O. Calculated: C 61.16%, H8.46%, N 4.09%, P 3.03%, Cl 3.41%. Found: C 61.21%, H 8.37%, N 4.04%, P2.98%, Cl 3.47%.

Example 3 Preparation of Lipid Derivatives of Ibuprofen (DP-Ibu)

[0285] The procedure for the preparation of lipid derivatives ofibuprofen (2-(4-isobutylphenyl)propionic acid) is the same as theprocess outlined in Example 1, steps 1 to 6, except that in step 6instead of diclofenac the drug included in the reaction mixture isibuprofen.

[0286] Lipid Derivatives of Ibuprofen (DP-Ibu)

[0287] The synthesized compounds were subjected to TLC analysis underthe following conditions: Silica gel 60 on aluminum sheet. Eluent ischloroform:methanol:water (65:35:5, v/v). Indicator is a spray of thecomposition: 4-methoxybenzaldehyde (10 ml), absolute ethanol (200 ml),98% sulfuric acid (10 ml) and glacial acetic acid (2 ml). Thechromatogram is sprayed with the indicator and then charred at 100° C.

[0288]1-Stearoyl-2-{3-[α-methyl-4-(2-methylpropyl)benzeneacetamido]propanoyl}-sn-glycero-3-phosphatidylcholine.

[0289] White wax. Hygroscopic. Yield 60%.

[0290] TLC analysis: One spot. R_(f) is 0.38.

[0291]¹H NMR (CD₃OD), δ (ppm): 0.88-0.93 (m, 9H), 1.29 (s, 28H),1.41-1.44 (d, 3H), 1.58-1.63 (m, 2H), 1.80-1.90 (m, 1H), 2.28-2.35 (t,2H), 2.43-2.46 (d, 2H), 2.51-2.57 (t, 2H), 3.22 (s, 9H), 3.40-3.45 (m,2H), 3.61-3.66 (m, 3H), 3.98-4.41 (several m, 6H), 5.18 (m, 1H),7.01-7.07 (d, 2H), 7.22-7.26 (d, 2H).

[0292]³¹P NMR (CD₃OD), δ (ppm): −0.20(s).

[0293] Chemical analysis: C₄₂H₇₅N₂O₉P.4H₂O. Calculated: C 59.02%, H9.93%, N 3.28%, P 3.63%. Found: C 59.26%, H 9.64%, N 3.43%, P 3.65%.

[0294]1-Stearoyl-2-{6-[α-methyl-4-(2-methylpropyl)benzeneacetamido]hexanoyl}-sn-glycero-3-phosphatidylcholine.

[0295] White wax. Hygroscopic. Yield 50%.

[0296] TLC analysis: One spot. R_(f) is 0.38.

[0297]¹H NMR (CD₃OD), δ (ppm): 0.88-0.93 (m, 9H), 1.29 (broad s, 31H),1.40-1.48 (m+d, 6H), 1.55-1.62 (m, 4H), 1.78-1.90 (m, 1H), 2.27-2.35 (m,4H), 2.43-2.46 (d, 2H), 3.11-3.16 (m, 2H), 3.22 (s, 9H), 3.56-3.66 (m,3H), 4.00-4.03 (t, 2H), 4.18-4.28 (several m, 4H), 5.18 (m, 1H),7.07-7.11 (d, 2H), 7.22-7.25 (d, 2H).

[0298]³¹P NMR (CD₃OD), δ (ppm): −0.20(s).

[0299] Chemical analysis: C₄₅H₈₁N₂O₉P.2.5H₂O. Calculated: C 62.07%, H9.89%, N 3.22%, P 3.56%. Found: C 62.00%, H 10.01%, N 3.32%, P 3.19%.

Example 4 Preparation of Lipid Derivatives of Naproxen (DP-Nap)

[0300] The procedure for the preparation of lipid derivatives ofnaproxen (d-2-(6-methoxy-2-naphthyl)propionic acid) is the same as theprocess outlined in Example 1, steps 1 to 6, except that in step 6instead of diclofenac the drug included in the reaction mixture isnaproxen.

[0301] Lipid Derivatives of Naproxen (DP-Nap)

[0302] The synthesized compounds were subjected to TLC analysis underthe following conditions: Silica gel 60 on aluminum sheet. Eluent ischloroform:methanol:water (65:35:5 v/v). Indicator is a spray of thecomposition: 4-methoxybenzaldehyde (10 ml), absolute ethanol (200 ml),98% sulfuric acid (10 ml) and glacial acetic acid (2 ml). Thechromatogram is sprayed with the indicator and then charred at 100° C.

[0303]1-Stearoyl-2-{3-[(S)-6-methoxy-α-methyl-2-naphtaleneacetamido]propanoyl}-sn-glycero-3-phosphatidylcholine.

[0304] White wax. Hygroscopic. Yield 65%.

[0305] TLC analysis: One spot. R_(f) is 0.38.

[0306]¹H NMR (CD₃OD), δ (ppm): 0.86-0.91 (t, 3H), 1.26 (s, 28H),1.50-1.53 (m, 5H), 2.23-2.29 (t, 2H), 2.51-2.57 (t, 2H), 3.16 (s, 9H),3.41-3.46 (t, 2H), 3.56-3.60 (m, 2H), 3.78-3.80 (m, 1H), 3.88 (s, 3H),3.97-4.02 (m, 2H), 4.12-4.31 (several m, 4H), 5.17-5.20 (m, 1H),7.08-7.13 (d, 1H), 7.19 (s, 1H), 7.41-7.45 (d, 1H), 7.70-7.73 (m, 3H).

[0307]³¹P NMR (CD₃OD), δ (ppm): −0.17(s).

[0308] Chemical analysis: C₄₃H₇₁N₂O₁₀P.4H₂O. Calculated: C 58.37%, H8.93%, N 3.17%, P 3.50%, Found: C 58.34%, H 8.98%, N 3.25%, P 3.58%.

[0309]1-Stearoyl-2-{4-[(S)-6-methoxy-α-methyl-2-naphtaleneacetamido]butanoyl}-sn-glycero-3-phosphatidylcholine.

[0310] White wax. Hygroscopic. Yield 65%.

[0311] TLC analysis: One spot. R_(f) is 0.38.

[0312]¹H NMR (CD₃OD), δ (ppm): 0.86-0.91 (t, 3H), 1.26 (s, 30H),1.50-1.53 (m, 5H), 1.73-1.82 (m, 2H), 2.26-2.33 (m, 4H), 3.17 (s, 11H),3.56-3.60 (m, 2H), 3.75-3.78 (m, 1H), 3.88 (s, 3H), 3.97-4.02 (m, 2H),4.12-4.31 (several m, 4H 5.17-5.20 (m, 1H), 7.08-7.13 (d, 1H), 7.19 (s,1H), 7.41-7.45 (d, 1H), 7.70-7.73 (m, 3H).

[0313]³¹P NMR (CD₃OD), δ (ppm): −0.22(s).

[0314] Chemical analysis: C₄₄H₇₃N₂O₁₀P.5H₂O. Calculated: C 58.10%, H9.12%, N 3.08%, P 3.40%. Found: C 58.69%, H 9.24%, N 3.18%, P 3.40%.

[0315]1-Stearoyl-2-{6-[(S)-6-methoxy-α-methyl-2-naphtaleneacetamido]hexanoyl}-sn-glycero-3-phosphatidyl

[0316] White wax. Hygroscopic. Yield 65%.

[0317] TLC analysis: One spot. R_(f) is 0.38.

[0318]¹H NMR (CD₃OD), δ (ppm): 0.87-0.91 (t, 3H), 1.26 (broad s, 32H),1.44-1.57 (several m, 10H), 2.23-2.31(m, 4H), 3.14-3.20 (s, 9H),3.60-3.62 (m, 2H), 3.75-3.78 (m, 1H), 3.88 (s, 3H), 4.00-4.02 (m, 2H),4.17-4.38 (several m, 4H), 5.22 (m, 1H), 7.09-7.12 (d, 1H), 7.19 (s,1H), 7.43-7.46 (d, 1H), 7.70-7.73 (m, 3H).

[0319]³¹P NMR (CD₃OD), δ (ppm): −0.80(s).

[0320] Chemical analysis: C₄₆H₇₇N₂O₁₀P.2H₂O. Calculated: C 62.44%, H9.16%, N 3.17%, P 3.50%. Found: C 62.73%, H 9.41%, N 3.29%, P 3.49%.

Example 5 Preparation of Lipid Derivatives of 6-methoxy-2-naphthylaceticacid (DP-MNap)

[0321] The procedure for the preparation of lipid derivatives of6-methoxy-2-naphthylacetic acid is the same as the process outlined inExample 1, steps 1 to 6, except that in step 6 instead of diclofenac thedrug included in the reaction mixture is 6-methoxy-2-naphthylaceticacid.

[0322] 6-methoxy-2-naphthylacetic acid was prepared according to theprocedure described by Khorana and Pishawikar (Indian J. Pharm. (1961)23: 297-301). Yield was 60%.

[0323] The final products, i.e. the phospholipid-drug conjugatesobtained at the end of step 6, were subjected to TLC analysis under thefollowing conditions: Silica gel 60 on aluminum sheet. Eluent ischloroform:methanol:water (65:35:5 v/v). Indicator is a spray of thecomposition: 4-methoxybenzaldehyde (10 ml), absolute ethanol (200 ml),98% sulfuric acid (10 ml) and glacial acetic acid (2 ml). Thechromatogram is sprayed with the indicator and then charred at 100° C.

[0324] A lipid derivative of 6-methoxy-2-naphthylacetic acid (DP-MNap)is depicted bellow.

[0325]1-Stearoyl-2-{4-[2-(6-methoxynaphtyl)acetamido]butanoyl}-sn-glycero-3-phosphatidylcholine.

[0326] White solid. Yield 60%.

[0327] TLC analysis: One spot. R_(f) is 0.5.

[0328]¹H NMR (CD₃OD), δ (ppm): 0.87-0.92 (t, 3H), 1.27 (broad s, 28H),1.53-1.56 (broad m, 2H), 1.78-1.84 (m, 2H), 2.25-2.40 (m, 4H), 3.15 (s,9H), 3.21-3.27 (m, 2H), 3.54-3.58 (m, 2H), 3.62 (s, 2H), 3.90 (s, 3H),4.00-4.04 (m, 2H), 4.18-4.23 (m, 3H), 4.36-4.41 (d, 1H), 5.20-5.24 (m,1H), 7.09-7.14 (d, 1H), 7.21-7.22 (m, 1H), 7.36-7.40 (d, 1H), 7.67-7.75(m, 3H).

[0329]³¹P NMR (CD₃OD), δ (ppm): 0.83(s).

[0330] Chemical analysis: C₄₃H₇₁N₂O₁₀P.2H₂O. Calculated: C 61.28%, H8.90%, N 3.32%, P 3.68%. Found: C 61.42%, H 9.08%, N 3.48%, P 3.70%.

Example 6 Solubility Measurements of Lipid Derivatives of Diclofenac,Indomethacin, Ibuprofen, Naproxen and 6-methoxy-2-naphthylacetic Acid

[0331] The solubility of several lipid derivatives of diclofenac,indomethacin, ibuprofen, naproxen and 6-methoxy-2-naphthylacetic acidwas determined at room temperature (22° C.) in aqueous solutions (waterand saline) and in organic solutions (ethanol and octanol). In addition,the partition coefficient (P_(c)) values for these compounds, i.e theirdistribution ratios between the organic and the aqueous phases(octanol/saline), were calculated. The results, presented as thecalculated LogP_(c), are shown in Table 1.

[0332] The distribution of the compounds between the organic and theaqueous phases was measured by the shake-flask technique. Fivemilliliters of octanol containing about 3 mg of the studied compoundwere mixed with 5 ml saline. The mixture was shaken overnight at 22° C.before the octanol and saline phases were separated. One ml from eachphase was dissolved into an appropriate volume of ethanol so that theoptical absorption of the obtained solution is in range of 0.1 to 1.0.

[0333] The coefficient P_(c) is calculated by the following ratio:$P_{c} = \frac{A_{oct}V_{1}l_{2}}{A_{sal}V_{2}l_{1}}$

[0334] where A_(oct) is the optical absorption, at λ_(max), of theethanol solution in which one ml of octanol phase is dissolved, V₁ isthe volume of this ethanol solution and l₁ is the width (cm) of thecuvette used for measurement of the optical absorption.

[0335] A_(sal) is the optical absorption, at λ_(max), of the ethanolsolution in which the one ml of saline phase is dissolved, V₂ is thevolume of this ethanol solution and l₂ is the width (cm) of the cuvetteused for measurement of the optical absorption.

[0336] Solubility values of the lipid derivatives of diclofenac,indomethacin, ibuprofen, naproxen and 6-methoxy-2-naphthylacetic acid inwater and saline and their octanol/saline distributions are presentedhereinbelow in table 1.

[0337] The tested lipid derivatives form suspensions or gel mixtures inwater and saline. Solutions containing the lipid derivatives formsuspensions or gel mixtures even after being filtrated through 0.45 μmfilter.

[0338] Table 1. Water and Saline Solubility and Octanol/SalineDistribution Coefficient (log P_(c)) of Lipid Derivatives at 22° C.

[0339] Lipid derivatives have the structure: lyso-lecithin-linker-drugwherein the linker is —C(O)—(CH₂)_(n)—NH—, the drug is diclofenac (DCF),indomethacin (Indo), ibuprofen (Ibu), naproxen (Nap) or6-methoxy-2-naphthylacetic acid (MNap). Lipid derivativeSolubility(mg/ml) Drug n Water Saline log P_(c) DCF 2 0.003^(a))0.002^(a)) 2.8 ± 0.1 DCF 3 0.002^(a)) 0.002^(a)) ≧3 DCF 4 0.002^(a))0.002^(a)) ≧3 DCF 5 0.0015^(b)) 0.0015^(b)) ≧3 DCF 7 0.001^(b))0.001^(b)) ≧3 DCF 11 <0.001^(b)) <0.001^(b)) ≧2.5 Indo 2 less than0.9^(a)) about 1.5^(a)) 2.5 Indo 3 less than 0.6^(a)) about 1.5^(a)) 2.5Indo 4 less than 0.01^(b)) about 0.1^(b)) 2.9 Indo 5 less than 0.01^(b))about 0.01^(b)) 3.2 Indo 7 about 0.005^(b)) — 3.7 Ibu 2 0.2^(a))0.3^(a)) 1.8 Ibu 5 0.5^(a)) 0.5^(a)) ≧3 Nap 2 0.01^(a)) 0.06^(a)) ≧3 Nap3 0.12^(a)) 0.09^(a)) ≧3 Nap 5 0.2^(a)) 0.2^(a)) ≧3 MNap 3 0.18^(a))0.14^(a)) ≧2.5

[0340] The solubility measurements indicate that all the lipidderivatives examined dissolve well in ethanol and octanol, i.e more than10 mg/ml at room temperature. The resulted solutions are transparent andthe compounds remain stable in the solution for at least several days atroom temperature.

[0341] It is evident that the lipid derivatives according to theinvention have acquired the desired hydrophobic properties that areadvantageous for brain penetration and sequestration.

Example 7 In Vitro Cleavage of DP-DFC in Tissue Homogenates

[0342] The ability of the compounds of the invention to be cleaved toyield free diclofenac, was studied in vitro in homogenates of rat brainand liver. Two compounds were tested: the prodrug1-Stearoyl-2-{4-[2′-(2″,6″-dichloroanilino)-phenylacetamido]butanoyl}-sn-glycero-3-phosphatidylcholine(DP-DFC; Z=3), and the compound1-Stearoyl-2-{3-[2′-(2″,6″-dichloroanilino)-phenylacetamido]propanoyl}-sn-glycero-3-phosphatidylcholine(DP-DFC; Z=2), comprising, respectively, a bridging group having a totalof 4 and 3 carbon atoms.

[0343] Liver and brain were surgically removed, under pentobarbitoneanesthesia, from male Sabra rats weighing 250-280 g. About 1-1.5 gsamples from each tissue were homogenized in PBS pH=7.4 (Dulbecco) at aratio of 1:9 w/v of tissue to buffer.

[0344] DP-DFC was added to each homogenate to a final concentration of40 μg/ml. The mixtures were incubated at 37° C. in shaking for 1, 2, 3and 6 hours, and then were placed on ice to stop the reaction.

[0345] The amounts of free diclofenac in 1 ml samples from either thebrain or liver homogenates were determined by reverse phase HPLC assay.The amount of protein in each sample was assayed by the Lowry method.

[0346] The amounts of free diclofenac released at each time point,calculated per 1 mg of protein, are shown in Table 2. The result foreach time point represents the average of five repetitions using tissuesfrom five individual animals. TABLE 2 In vitro cleavage of DP-DFC FreeDFC (μg/mg protein) DP-DFC; Z = 3 DP-DFC; Z = 2 Time (hrs) brain liverbrain liver 0 0.05 0.07 N.D N.D 1 0.36 ± 0.08 0.18 ± 0.09 N.D N.D 2 0.65± 0.06 0.23 ± 0.05 N.D N.D 3 0.75 ± 0.15 0.33 ± 0.14 N.D N.D 6 1.30 ±0.40 0.45 ± 0.14 N.D N.D

[0347] As shown in Table 2, a significant cleavage of diclofenac fromits lipid conjugate was demonstrated, both in the brain and liverhomogenates, only for the prodrug DP-DFC; Z=3 and not for the compoundDP-DFC; Z=2. These results assess the important role the bridging groupplays in enabling the release of the drug from its phospholipidderivative.

[0348] Under the above-described experimental conditions, the amounts ofdiclofenac released from the prodrug in the brain and liver homogenateswere equivalent, respectively, to 30% and 15% of the drug introduced asDP-DFC.

[0349] In order to evaluate the effect of temperature on the reaction, aparallel set of incubations was carried out at 4° C. It was found thatcooling to 4° C. completely inhibited the cleavage of DP-DFC; Z=3 to DFC(data not shown).

[0350] The time and temperature dependent manner of the diclofenacrelease in the homogenates supports the conclusion that the drug isenzymatically cleaved from the prodrug molecule.

Example 8 In Vivo Sub-Chronic Toxicity Study of DP-DCF

[0351] DP-DCF compounds were evaluated for sub-chronic toxicity during14-day drug administration period by following two criteria: a)monitoring body weight changes, and b) evaluating gastro-toxicityscores.

[0352] Male Sprague-Dawley rats weighing 250-280 g were kept understandard conditions for an acclimatization period of one week with foodand water supplied ad lib. The rats were administered with commercialdiclofenac (DCF; Sigma, USA) or with equivalent doses of1-Stearoyl-2-{4-[2′-(2″,6″-dichloroanilino)-phenylacetamido]butanoyl}-sn-glycero-3-phosphatidylcholine(DP-DCF; Z=3) or1-Stearoyl-2-{6-[2′-(2″,6″-dichloroanilino)-phenylacetamido]hexanoyl}-sn-glycero-3-phosphatidylcholine(DP-DCF; Z=5), p.o. by gavage daily for 14 days. Animals treated withthe vehicle solution alone serve as control group.

[0353] The treatment groups were as follows: Group No. of animalsTreatment Dosage (mg/kg) 1 5 vehicle (control) — 2 5 DCF 10 3 5 DP-DCF;Z = 3 30 4 5 DP-DCF; Z = 5 30

[0354] A. Body Weight Changes

[0355] A major sign of general toxicity is decreasing of body weight.Male Sabra rats were treated as described above. The rats were weighedprior to dosing at the beginning of the experiment (day zero) and at the5^(th), 9^(th) and 13^(th) day of the treatment. The percentage changesof body weight are shown in FIG. 1.

[0356] As can be seen in FIG. 1, DCF treated animals exhibit maximaldecrease of body weight on day 5 of the experiment. In contrast to DCFtreated animals, all the DP-DCF treated groups gained weight over 14days with a similar profile to the vehicle treated group.

[0357] B. Toxicity Scores

[0358] Toxicity scores were assessed at the end of the 14-day multiplep.o drug administration period. On day 14, the animals were anesthetized4 hours after administration of the test materials by i.p. injection of0.5 ml pentobarbitone sodium (200 mg/ml) and laparatomy was preformed toremove the rat's stomach with approximately 15 mm of the associatedduodenum. The stomachs were cut open along the small curvature andwashed, with gentle rubbing, under running water. A semi-quantitativeassessment of the gastric damage was determined by assigning scoresranging from 0 to 5. The scores indicate as follows: (0) Nomacroscopically visible lesions; (1) Hyperemia; (2) One-two slightlesions; (3) More than 2 slight lesions; (4) Severe lesions; (5)Perforation.

[0359] As shown in FIG. 2, the DP-DCF derivatives were less toxic thanthe parent drug, DCF. Both tested compounds, DP-DCF; Z=3 and DP-DCF;Z=5, show a better toxicological profile than diclofenac. The gastricside effects of DCF increase during multiple dosage administration andtoxicity scores of 4 were reached by day 14. The irritant effects of thetested DP-DCF compounds were similar to that obtained with the vehicle,namely toxicity scores between 0.25 and 1.

[0360] Conclusion: As assessed by two parameters monitored during a2-week study, the tested DP-DCFs were found to be substantially lesstoxic than the parent compounds. Normal weight gain was observed overtwo weeks in the DP-DCF treated animals compared with weight lossesobserved with the parent compound, diclofenac. The compounds of theinvention were also shown to be less ulcerogenic as judged by thegastro-toxicity scores obtained after 14 days of chronic p.o. drugadministration.

Example 9 In Vivo Penetration of Diclofenac into Rat Brain after i.v.Injection of DP-DFC

[0361] The time-dependent penetration of lipid conjugates of diclofenacis measured in rat serum and brain after i.v. administration of DP-DFC.10 mg/kg of DP-DFC are intravenously injected to male Sabra ratsweighing 250-280 g. The prodrug (5 mg/ml) is formulated as follows: 50mg DP-DFC are dissolved in 300-400 μl ethanol, and Lipofundin® (B.Braun, Melsungen, Germany) is added upto a final volume of 10 ml. Theamount of the formulated prodrug injected to the animals is 2 ml/kg bodyweight.

[0362] The animals are sacrificed either 0.5, 1, 2 or 3 hours followinginjection. Whole brain and blood samples are obtained from individualrats for each time point. The serum is separated by centrifugation ofthe blood (5 min. at about 800×g). The brain is treated as follows:whole brain is homogenized in saline. Half of the homogenate isextracted into organic solvents (methanol-chloroform 1:2) and is usedfor determination the level of the lipid derivative (DP-DFC). The otherhalf is acidified by 85% H₃PO₄, extracted into chloroform and is usedfor determination of the level of free diclofenac (DFC). Each of theorganic phases is separated by centrifugation, dried over Na₂SO₄ andevaporated. The obtained residues are dissolved in the correspondingmobile phase used in the HPLC method, and the amounts of both DP-DFC andDFC are determined by reverse phase HPLC.

[0363] Conclusion: Following i.v. administration of the prodrug, DP-DFCpenetrates the blood-brain barrier (BBB).

Example 10 Anti-Inflammatory Activity of DP-DFC (In Vivo Efficacy Study)

[0364] Efficacy of DP-DFC lipid derivatives was tested in vivo in themodel system of rodent carrageenan edema test.

[0365] Carrageenan-induced rat paw edema is a widely employed animalmodel for acute inflammation. The objective of the study is to assessthe potential prophylactic effects of DP-DCF derivatives on theprevention of inflammatory swelling and, in particular, to compare theefficacy parameters with those obtained for diclofenac. The experimentalset-up was as follows: Male Sprague-Dawley rats weighing 120-180 g(supplied by Harlan Laboratories Breeding Center, Israel) wereintraperitoneally (i.p.) injected with drug or prodrug one hour prior tothe induction of inflammation with carrageenan. Animals injected withvehicle alone served as control group.

[0366] Paw edema was induced by a single sub-plantar injection of 0.1 ml2% carrageenan in physiological saline, into test animals' right hindpaws. Just prior to paw edema induction, the paw thickness of the testanimals' right hind paws was measured in triplicate using aplethysmograph and micrometer to provide a baseline. At 3, 5 and 7 hourspost carrageenan injection, the right paw thickness was measured in thesame manner as before.

[0367] Potential anti-inflammatory activity in suppressingcarrageenan-induced paw oedema was assessed by the relative differencesin paw volumes between pre- and post-carrageenan measurements, expressedas (%) change.

[0368] Dose levels were 10 mg/kg i.p. for diclofenac and 30 mg/kg (doseequivalent) for the DP-DCF derivatives. 10 ml/kg of the carrier solutionwas used for the vehicle control.

[0369] In FIG. 3 are presented the results obtained with one of theDP-derivatives, DP-DCF; Z=5, in comparison to those obtained with theparent compound, diclofenac. As can be seen in FIG. 3, both DCF and theDP-DCF derivative demonstrated statistically significant and consistentanti-inflammatory activity throughout the 7-hour post-carrageenanmeasurement period (level of significance is p<0.05 by statisticalanalysis using the Student's t-test). Anti-inflammatory activity wasalready found at the 3-hour post-carrageenan measurement.

Example 11

[0370] Neuroprotective Effects of DP-DCF

[0371] Neuroprotective effects of compounds of the invention wereexamined in the global forebrain ischemic model system in Mongoliangerbils.

[0372] Male Mongolian gerbils 60-70 g (Charles River Laboratories, USA)were used in this study. Global forebrain ischemia was induced bytransient occlusion of both carotid arteries for 10 minutes, usingarterial clips. The tested compounds were orally administered to theanimals, 7-8 animals per group, by either one of two schemes:

[0373] a) single dose administration of commercial diclofenac (Sigma,USA; 10 mg/kg) or dose-equivalent of DP-DCF; Z=5 (30 mg/kg). Theadministration was per os (p.o.) two hours before induction of ischemia(FIG. 4);

[0374] b) multiple dosing of DP-DCF; Z=5 (30 mg/kg) orally administereddaily for 5 days beginning four days prior to induction of ischemia.(FIG. 5).

[0375] In both schemes, animals treated with vehicle alone served ascontrol groups. Blood samples were withdrawn from eye sinuses of theanimals 24 hours after the onset of ischemia and serum levels of neuronspecific enolase (NSE) were determined by radioimmunoassay using NSE kit(Pharmatope Ltd., Israel). The measured NSE levels, expressed in ng/ml,are summarized in FIGS. 4 and 5.

[0376] It has been shown that serum levels of NSE are elevated in theevent of ischemic insult. Thus, serum level of NSE may serve as a markerfor the degree of neuronal damage (Barone et al. (1993) Brain Res. 623:77-82).

[0377] As shown in FIG. 4, NSE level in serum of animals treated withsingle dose of DP-DCF is reduced in comparison to NSE level in serum ofanimals that were treated with DCF or vehicle alone. The parent drug,diclofenac, penetrates the brain poorly and is not expected to have aneffect on NSE level changes associated with brain ischemia.

[0378] As shown in FIG. 5, which summarizes the results of the multipledosing experiment, also in this case there is significant reduction inNSE level found in the serum of animals treated with the DP-DCF compound(p<0.05).

[0379] Conclusion: The tested DP-DCF derivative exhibits neuronalprotective effect, as indicated by the significant reduction in NSEactivity in the serum, measured after global cerebral ischemia. Theeffect was demonstrated in both single and multiple dosing schemes.

[0380] While the present invention has been particularly described,persons skilled in the art will appreciate that many variations andmodifications can be made. Therefore, the invention is not to beconstrued as restricted to the particularly described embodiments,rather the scope, spirit and concept of the invention will be morereadily understood by reference to the claims which follow.

1. A compound of the general formula I

or a pharmaceutically acceptable salt thereof, wherein: R1 is asaturated or unsaturated, substituted or unsubstituted hydrocarbon chainhaving from 2 to 30 carbon atoms; R2 is H or a phospholipid head group;D is the residue of a nonsteroidal anti-inflammatory drug having afunctional group selected from the group consisting of carboxyl,hydroxyl, amine and thiol, wherein D is attached through said functionalgroup to a bridging group, —C(O)-Z-X—, wherein Z is a saturated orunsaturated hydrocarbon chain having from 2 to 15 carbon atoms, and X isselected from amino, hydroxy, thio and carbonyl groups, such that whenthe functional group of D is carboxyl, X is selected from amino, hydroxyand thio, and when the functional group of D is amino, hydroxy or thio,X is a carbonyl group.
 2. The compound according to claim 1, wherein thedrug derivative is inactive.
 3. The compound according to claim 1,wherein an ester bond at position sn-2 of the phospholipid of thegeneral formula I is cleaveable by a lipase.
 4. The compound accordingto claim 3, wherein said lipase is a phospholipase.
 5. The compoundaccording to claim 4, wherein said phospholipase is phospholipase A₂(PLA₂).
 6. The compound according to claim 1, wherein R1 is anhydrocarbon chain having from 10 to 20 carbon atoms.
 7. The compoundaccording to claim 1, wherein R1 is an hydrocarbon chain having 15 or 17carbon atoms.
 8. The compound according to claim 1, wherein D isselected from the group consisting of diclofenac, indomethacin,ibuprofen, naproxen and 6-methoxy-2-naphthylacetic acid.
 9. The compoundaccording to claim 1, wherein R2 is selected from the group consistingof choline, ethanolamine, inositol and serine.
 10. The compoundaccording to claim 1 selected from the group consisting of:1-Stearoyl-2-{3-[2′-(2″,6″-dichloroanilino)phenylacetamido]propanoyl}-sn-glycero-3-phosphatidylcholine,1-Stearoyl-2-{4-[2′-(2″,6″-dichloroanilino)phenylacetamido]butanoyl}-sn-glycero-3-phosphatidylcholine,1-Stearoyl-2-{5-[2′-(2″,6″-dichloroanilino)phenylacetamido]valeroyl}-sn-glycero-3-phosphatidylcholine,1-Stearoyl-2-{6-[2′-(2″,6″-dichloroanilino)phenylacetamido]hexanoyl}-sn-glycero-3-phosphatidylcholine,1-Stearoyl-2-{8-[2′-(2″,6″-dichloroanilino)phenylacetamido]octanoyl}-sn-glycero-3-phosphatidylcholine,1-Stearoyl-2-{12-[2′-(2″,6″-dichloroanilino)phenylacetamido]dodecanoyl}-sn-glycero-3-phosphatidylcholine,1-Stearoyl-2-{3-[1-(p-chlorobenzoyl)-5-methoxy-2-methylindolylacetamido]propanoyl}-sn-glycero-3-phosphatidylcholine,1-Stearoyl-2-{4-[1-(p-chlorobenzoyl)-5-methoxy-2-methylindolylacetamido]butanoyl}-sn-glycero-3-phosphatidylcholine,1-Stearoyl-2-{5-[1-(p-chlorobenzoyl)-5-methoxy-2-methylindolylacetamido]valeroyl}-sn-glycero-3-phosphatidylcholine,1-Stearoyl-2-{6-[1-(p-chlorobenzoyl)-5-methoxy-2-methylindolylacetamido]hexanoyl}-sn-glycero-3-phosphatidylcholine,1-Stearoyl-2-{8-[1-(p-chlorobenzoyl)-5-methoxy-2-methylindolylacetamido]octanoyl}-sn-glycero-3-phosphatidylcholine,1-Stearoyl-2-{3-[α-methyl-4-(2-methylpropyl)benzeneacetamido]propanoyl}-sn-glycero-3-phosphatidylcholine,1-Stearoyl-2-{6-[α-methyl-4-(2-methylpropyl)benzeneacetamido]hexanoyl}-sn-glycero-3-phosphatidylcholine,1-Stearoyl-2-{3-[(S)-6-methoxy-α-methyl-2-naphtaleneacetamido]propanoyl}-sn-glycero-3-phosphatidylcholine,1-Stearoyl-2-{4-[(S)-6-methoxy-α-methyl-2-naphtaleneacetamido]butanoyl}-sn-glycero-3-phosphatidylcholine,1-Stearoyl-2-{6-[(S)-6-methoxy-α-methyl-2-naphtaleneacetamido]hexanoyl}-sn-glycero-3-phosphatidylcholine,and1-Stearoyl-2-{4-[2-(6-methoxynaphtyl)acetamido]butanoyl}-sn-glycero-3-phosphatidylcholine.11. A pharmaceutical composition comprising a pharmaceuticallyacceptable carrier and, as an active ingredient, a compound of thegeneral formula I

or a pharmaceutically acceptable salt thereof, wherein: R1 is asaturated or unsaturated, substituted or unsubstituted hydrocarbon chainhaving from 2 to 30 carbon atoms; R2 is H or a phospholipid head group;D is the residue of a nonsteroidal anti-inflammatory drug having afunctional group selected from the group consisting of carboxyl,hydroxyl, amine and thiol, wherein D is attached through said functionalgroup to a bridging group, —C(O)-Z-X—, wherein Z is a saturated orunsaturated hydrocarbon chain having from 3 to 15 carbon atoms, and X isselected from amino, hydroxy, thio and carbonyl groups, such that whenthe functional group of D is carboxyl, X is selected from amino, hydroxyand thio, and when the functional group of D is amino, hydroxy or thio,X is a carbonyl group.
 12. The pharmaceutical composition according toclaim 11, wherein —C(O)-Z-X-D is an inactive drug derivative.
 13. Thepharmaceutical composition according to claim 11, wherein an ester bondat position sn-2 of the phospholipid of the general formula I iscleaveable by a lipase.
 14. The pharmaceutical composition according toclaim 13, wherein said lipase is a phospholipase.
 15. The pharmaceuticalcomposition according to claim 14, wherein said phospholipase isphospholipase A₂ (PLA₂).
 16. The pharmaceutical composition according toclaim 11, wherein R1 is an hydrocarbon chain having from 10 to 20 carbonatoms.
 17. The pharmaceutical composition according to claim 11, whereinR1 is an hydrocarbon chain having 15 or 17 carbon atoms.
 18. Thepharmaceutical composition according to claim 11, wherein D is selectedfrom the group consisting of diclofenac, indomethacin, ibuprofen,naproxen and 6-methoxy-2-naphthylacetic acid.
 19. The pharmaceuticalcomposition according to claim 11, wherein R2 is selected from the groupconsisting of choline, ethanolamine, inositol and serine.
 20. Thepharmaceutical composition according to claim 11, wherein said compoundof the general formula I is selected from the group consisting of:1-Stearoyl-2-{3-[2′-(2″,6″-dichloroanilino)phenylacetamido]propanoyl}-sn-glycero-3-phosphatidylcholine,1-Stearoyl-2-{4-[2′-(2″,6″-dichloroanilino)phenylacetamido]butanoyl}-sn-glycero-3-phosphatidylcholine,1-Stearoyl-2-{5-[2′-(2″,6″-dichloroanilino)phenylacetamido]valeroyl}-sn-glycero-3-phosphatidylcholine,1-Stearoyl-2-{6-[2′-(2″,6″-dichloroanilino)phenylacetamido]hexanoyl}-sn-glycero-3-phosphatidylcholine,1-Stearoyl-2-{8-[2′-(2″,6″-dichloroanilino)phenylacetamido]octanoyl}-sn-glycero-3-phosphatidylcholine,1-Stearoyl-2-{12-[2′-(2″,6″-dichloroanilino)phenylacetamido]dodecanoyl}-sn-glycero-3-phosphatidylcholine,1-Stearoyl-2-{3-[1-(p-chlorobenzoyl)-5-methoxy-2-methylindolylacetamido]propanoyl}-sn-glycero-3-phosphatidylcholine,1-Stearoyl-2-{4-[1-(p-chlorobenzoyl)-5-methoxy-2-methylindolylacetamido]butanoyl}-sn-glycero-3-phosphatidylcholine,1-Stearoyl-2-{5-[1-(p-chlorobenzoyl)-5-methoxy-2-methylindolylacetamido]valeroyl}-sn-glycero-3-phosphatidylcholine,1-Stearoyl-2-{6-[1-(p-chlorobenzoyl)-5-methoxy-2-methylindolylacetamido]hexanoyl}-sn-glycero-3-phosphatidylcholine,1-Stearoyl-2-{8-[1-(p-chlorobenzoyl)-5-methoxy-2-methylindolylacetamido]octanoyl}-sn-glycero-3-phosphatidylcholine,1-Stearoyl-2-{3-[α-methyl-4-(2-methylpropyl)benzeneacetamido]propanoyl}-sn-glycero-3-phosphatidylcholine,1-Stearoyl-2-{6-[α-methyl-4-(2-methylpropyl)benzeneacetamido]hexanoyl}-sn-glycero-3-phosphatidylcholine,1-Stearoyl-2-{3-[(S)-6-methoxy-α-methyl-2-naphtaleneacetamido]propanoyl}-sn-glycero-3-phosphatidylcholine,1-Stearoyl-2-{4-[(S)-6-methoxy-α-methyl-2-naphtaleneacetamido]butanoyl}-sn-glycero-3-phosphatidylcholine,1-Stearoyl-2-{6-[(S)-6-methoxy-α-methyl-2-naphtaleneacetamido]hexanoyl}-sn-glycero-3-phosphatidylcholine,and1-Stearoyl-2-{4-[2-(6-methoxynaphtyl)acetamido]butanoyl}-sn-glycero-3-phosphatidylcholine.21. The pharmaceutical composition according to any one of claims 11 to20, in the form of solutions, suspensions, capsules, tablets, aerosols,gels, ointments or suppositories.
 22. The pharmaceutical compositionaccording to any one of claims 11 to 20 for oral, ocular, nasal,parenteral, topical or rectal administration.
 23. The pharmaceuticalcomposition according to claim 22 for oral administration.
 24. Thepharmaceutical composition according to claim 22 for nasaladministration.
 25. The pharmaceutical composition according to any oneof claims 11 to 24 for the treatment of a disease or disorder related toan inflammatory condition.
 26. The pharmaceutical composition accordingto claim 25, wherein said disease or disorder related to an inflammatorycondition is selected from the group consisting of arthritis, rheumatoidarthritis, asthma, psoriasis, systemic lupus erythematosus, inflammatorybowel syndrome and the neurological diseases and disorders multiplesclerosis, Alzheimer's disease, Parkinson's disease, Huntington'sdisease, vascular dementia, epilepsy, migraines, stroke and trauma. 27.Use for the manufacture of a medicament of a compound of the generalformula I

or a pharmaceutically acceptable salt thereof, wherein: R1 is asaturated or unsaturated, substituted or unsubstituted hydrocarbon chainhaving from 2 to 30 carbon atoms; R2 is H or a phospholipid head group;D is the residue of a nonsteroidal anti-inflammatory drug having afunctional group selected from the group consisting of carboxyl,hydroxyl, amine and thiol, wherein D is attached through said functionalgroup to a bridging group, —C(O)-Z-X—, wherein Z is a saturated orunsaturated hydrocarbon chain having from 3 to 15 carbon atoms, and X isselected from amino, hydroxy, thio and carbonyl groups, such that whenthe functional group of D is carboxyl, X is selected from amino, hydroxyand thio, and when the functional group of D is amino, hydroxy or thio,X is a carbonyl group.
 28. A method for treatment of a disease ordisorder related to an inflammatory condition comprising administeringto a patient in need thereof a therapeutically effective amount of apharmaceutical composition according to any one of claims 11 to
 26. 29.The method according to claim 28, wherein said disease or disorderrelated to an inflammatory condition is selected from the groupconsisting of arthritis, rheumatoid arthritis, asthma, psoriasis,systemic lupus erythematosus, inflammatory bowel syndrome and theneurological diseases and disorders multiple sclerosis, Alzheimer'sdisease, Parkinson's disease, Huntington's disease, vascular dementia,epilepsy, migraines, stroke and trauma.
 30. A process for the synthesisof compounds of the general formula I as defined in claim 1, comprising:(i) providing a molecule y-X-Z-COOH, wherein y is selected from H andOH, Z is a saturated or unsaturated hydrocarbon chain having from 2 tocarbon atoms, and X is selected from amino, hydroxy, thio and carbonylgroups; (ii) replacing y with an appropriate blocking group, B; (iii)preparing an anhydride of the molecule B-X-Z-COOH; (iv) acylating alyso-lecithin by the anhydride of step (iii) to yield1-acyl-2-acyl(X-B)-sn-glycero-3 phospholipid; (v) removing the blockinggroup B from the functional group X; and (vi) coupling a nonsteroidalanti-inflammatory drug D to the functional group X, thus, generating amolecule of the general Formula I.
 31. The process according to claim 30wherein the protected functional group X is —NH.
 32. The processaccording to claim 30 wherein the phospholipid of step (iv) isphosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol orphosphatidylserine.
 33. The process according to claim 30 wherein thenonsteroidal anti-inflammatory drug D is selected from the groupconsisting of diclofenac, indomethacin, ibuprofen, naproxen and6-methoxy-2-naphthylacetic acid.